Methods and compositions for vaccination comprising nucleic acid and/or polypeptide sequences of Chlamydia psittaci

ABSTRACT

The instant invention relates to antigens and nucleic acids encoding such antigens obtainable by screening the  Chlamydia psittaci  genome. In more specific aspects, the invention relates to methods of isolating such antigens and nucleic acids and to methods of using such isolated antigens for producing immune responses in bovines or other non-human animals. The ability of an antigen to produce an immune response may be employed in vaccination of bovines or antibody preparation techniques.

[0001] The government owns rights in the present invention pursuant toDARPA grant number MDA 972-97-1-0013.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields ofimmunology, bacteriology and molecular biology. More particularly, theinvention relates to methods for screening and obtaining vaccinesgenerated from the administration of expression libraries constructedfrom a Chlamydia psittaci genome. In particular embodiments, it concernsmethods and compositions for the vaccination of vertebrate animalsagainst Chlamydia psittaci bacterial infections, wherein vaccination ofthe animal is via a protein or gene derived from part or all of thegenes validated as vaccines.

[0004] 2. Description of Related Art

[0005] Intracellular bacteria of the genus Chlamydia are importantpathogens in both man and vertebrate animals, causing blindness in man,sexually transmitted disease, and community-acquired pneumonia, and mostlikely act as co-factors in atherosclerotic plaque formation in humancoronary heart disease.

[0006] Ubiquitous Chlamydia (C) psittaci infections in cattle causemastitis, infertility and abortion. A primary economic impact ofChlamydia psittaci in dairy cattle is the loss of milk production andquality. Serological evidence for infection with ruminant C. psittaci isfound in virtually all cattle (Kaltenbock et al., 1997). Theseinfections typically do not cause overt signs of disease, but understress of the host animal may elicit transient inflammation of themammary gland and uterus. These stress-related herd health problems,while not clinically pronounced, result in major losses for animalagriculture due to reduced output and quality of animal products likemilk.

[0007] Most existing vaccines for the treatment of bacterial infectionsare composed of live/attenuated or killed pathogens (Babiuk, 1999).Live/attenuated vaccines present the risk of residual, or reacquisitionof, pathogenicity, and are associated with a high cost of production. Inaddition, efficacious live/attenuated vaccines cannot be developedagainst many pathogens, or are impractical to produce. Killed pathogenstypically have less utility than live/attenuated vaccines, as they arenot usually effective in eliciting cellular immune responses. Analternative is subunit vaccines that consist of one or a few proteins ofthe pathogen (Babiuk, 1999; Ellis, 1999). The proteins being developedfor these vaccines are typically based on a dominant immune response ininfected hosts, and/or on surmised importance in the disease process.Due to the high genetic complexity of bacteria or protozoa, theempirical approach to identify these proteins often requires extensiveresearch on the pathogen's biology and produces a small, biased set ofpotential vaccine candidates. However, this is currently the onlypractical method when proteins are the commodity for testing a vaccine.

[0008] The development of genetic (DNA) immunization (Tang et al., 1992)not only offers a new method of vaccine delivery, but also enables anew, unbiased, approach to vaccine discovery. One of the inventorsproposed that the whole genome of a pathogen could be searched forprotein vaccine candidates by directly assessing protection fromchallenge, termed expression library immunization (ELI) (U.S. Pat. No.5,703,057, specifically incorporated herein by reference). It involvesmaking an expression library representing the whole genome of thepathogen in a genetic immunization vector. The library is subdividedinto smaller groups, and DNA from each library is used to vaccinateanimals that are subsequently challenged. The advantage of this approachis that all of the potentially protective genes could be discovered andused in any useful combination to reconstitute a vaccine devoid ofnon-protective, immunopathological, or immunosuppressive antigens. Thepotential of ELI was demonstrated in a murine Mycoplasma pulmonisinfection, against which random M. pulmonis libraries were protective(Barry et al., 1995). Since then, others have reported on protectivelibraries (Brayton et al., 1998; Piedrafita et al., 1999), but thereduction of these libraries to individual genes has not beendemonstrated.

[0009] As described above, the widespread human and animal infections bythe genus Chlamydia psittaci represents a particular challenge forvaccinology. C. psittaci infections in cattle cause mastitis,infertility and abortion. A primary economic impact of Chlamydiapsittaci in dairy cattle is the loss of milk production and quality.Thus, an effective vaccine against Chlamydia psittaci bacterialinfections in cattle would be of great economic importance. However,there presently have been no effective vaccines developed againstChlamydia psittaci.

SUMMARY OF THE INVENTION

[0010] In some embodiments, the invention relates to isolatedpolynucleotides having a region that comprises a sequence of SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:58, or SEQ ID NO:60, a complement of anyof these sequences or fragments thereof. In some more specificembodiments, the invention relates to such polynucleotide comprising aregion having a sequence comprising at least 17, 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 100, 125, 150, 200, or more contiguous nucleotidesin common with at least one of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24,SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44,SEQ ID NO:46, SEQ ID NO:58, or SEQ ID NO:60, or its complement. Ofcourse, such polynucleotides may comprise a region having allnucleotides in common with at least one of SEQ ID NO:6, SEQ ID NO:8, SEQID NO:δ0, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO: 18, SEQID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:58, or SEQ ID NO:60, or its complement.

[0011] In other aspect, the invention relates to polypeptides havingsequences of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:59, SEQ ID NO:61 or fragments thereof. The invention also relates tomethods of producing such polypeptides using recombinant methods, forexample, using the polynucleotides described above.

[0012] The invention relates to antibodies against Chlamydia psittaciantigens, including those directed against an antigen having sequencesof SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:61, or an antigenic fragmentthereof. The antibodies may be polyclonal or monoclonal and produced bymethods known in the art.

[0013] The invention relates to vaccines for the immunization of bovinesagainst Chlamydia psittaci. Such vaccines may comprise apharmaceutically acceptable carrier, and at least one polynucleotidehaving a Chlamydia psittaci sequence. Such vaccines may comprise atleast one polynucleotide that has a sequence isolated from a Chlamydiapsittaci genomic DNA expression library. In some preferred embodiments,the vaccine comprises at least one polynucleotide having a sequence ofSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO52:, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO: 58, or SEQ ID NO:60, or fragment thereof. In somespecific preferred embodiments, the vaccine comprises at least onepolynucleotide having a sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, or SEQ ID NO:26, or fragment thereof, while in someeven more specific embodiments, the vaccine comprises at least onepolynucleotide having a sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:20, or SEQ ID NO:24. The polynucleotide may becomprised in a genetic immunization vector. Some vectors useful in theinvention comprise a gene encoding a mouse ubiquitin fusion polypeptideand/or a promoter operable in eukaryotic cells, for example a CMVpromoter. The polynucleotide may be cloned into a viral expressionvector, for example, a viral expression vector selected from the groupconsisting of adenovirus, adeno-associated virus, retrovirus andherpes-simplex virus.

[0014] In some embodiments, the vaccine comprises at least a firstpolynucleotide having a Chlamydia psittaci sequence and a secondpolynucleotide having a Chlamydia psittaci sequence, wherein the firstpolynucleotide and the second polynucleotide have different Chlamydiapsittaci sequences. In some preferred embodiments, the firstpolynucleotide has a sequence of SEQ ID NO:50.

[0015] Other embodiments of vaccines for the immunization of a bovineagainst Chlamydia psittaci comprise a pharmaceutically acceptablecarrier and at least one Chlamydia psittaci antigen. The at least oneChlamydia psittaci antigen can be an antigen having a sequence of SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, or SEQ ID NO:61, or an antigenic fragment thereof.In some specific embodiments, the vaccine comprises at least oneChlamydia psittaci antigen having a sequence of SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27 or an antigenicfragment thereof. In some even more specific embodiments, the at leastone Chlamydia psittaci antigen has a sequence of SEQ ID NO:7, SEQ IDNO:11, SEQ ID NO:15, SEQ ID NO:21, or SEQ ID NO:25.

[0016] The invention contemplates methods of immunizing a bovinecomprising providing to the bovine at least one Chlamydia psittaciantigen, or antigenic fragment thereof, in an amount effective to inducean immune response. The antigens described above are examples ofparticularly useful antigens in this regard. The provision of at leastone Chlamydia psittaci antigen may comprise: (a) preparing a clonedexpression library from fragmented genomic DNA, cDNA or sequenced genesof Chlamydia psittaci; (b) administering at least one clone of thelibrary in a pharmaceutically acceptable carrier into the bovine; and(c) expressing at least one Chlamydia psittaci antigen in the bovine.The polynucleotide may be administered, for example, by a intramuscularinjection or epidermal injection or intravenous, subcutaneous,intralesional, intraperitoneal, oral or inhaled routes ofadministration. An intramuscular injection may comprise least 1.0 μg to200 μg of the polynucleotide, whereas an epidermal injection maycomprise at least 0.01 μg to 5.0 μg of the polynucleotide. Secondintramuscular injection or epidermal injections may be administered, forexample, at least about three weeks after the first injection. Thepolynucleotide may be comprised in a viral expression vector.

[0017] Alternatively, the provision of the Chlamydia antigen(s) maycomprise: (a) preparing a pharmaceutical composition comprising at leastone polynucleotide having a sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO52:, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, or SEQID NO:60, or fragment thereof; (b) administering one or more clones ofthe library in a pharmaceutically acceptable carrier into the bovine;and (c) expressing one or more Chlamydia antigens in the bovine. Theantigen may be administered in much the same manner as described forpolynucleotides above, and other manners known to those of skill in theart.

[0018] In another alternative, the provision of the Chlamydia antigen(s)may comprise: (a) preparing a pharmaceutical composition of at least oneChlamydia antigen having a sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ IDNO:61, or an antigenic fragment thereof; and (b) administering the atleast one antigen or fragment into the animal.

[0019] This specification discloses methods of obtaining polynucleotidesequences effective for generating an immune response against Chlamydiapsittaci comprising: (a) preparing a cloned expression library fromfragmented genomic DNA of the species Chlamydia psittaci; (b)administering one or more clones of the library in a pharmaceuticallyacceptable carrier into the animal in an amount effective to induce animmune response; and (c) selecting from the library the polynucleotidesequences that induce an immune response, wherein the immune response inthe animal is protective against Chlamydia psittaci infection. Suchmethods may further comprise testing the animal for immune resistanceagainst a Chlamydia psittaci bacterial infection by challenging theanimal with Chlamydia psittaci. The genomic DNA is fragmented physicallyor by restriction enzymes, and in some preferred embodiments, thefragments are about 200-1000 base pairs. In some cases each clone in thelibrary may comprise a gene encoding a mouse ubiquitin fusionpolypeptide designed to link the expression library polynucleotides tothe ubiquitin gene. In some embodiments, the library is about 1×10³ toabout 1×10⁶ clones, with some preferred embodiments using a libraryhaving 1×10⁵ clones. In some cases, about 0.01 μg to about 200 μg ofDNA, cDNA or sequenced gene from the clones is administered into theanimal, for example by intramuscular injection or epidermal injection.In many cases, the cloned expression library further comprises apromoter operably linked to the DNA that permits expression in avertebrate animal cell.

[0020] The invention also relates to methods of assaying for thepresence of Chlamydia psittaci infection in a bovine comprising: (a)obtaining an antibody directed against a Chlamydia psittaci antigen; (b)obtaining a sample from the bovine; (c) admixing the antibody with thesample; and (d) assaying the sample for antigen-antibody binding,wherein the antigen-antibody binding indicates Chlamydia psittaciinfection in the bovine. In many cases, the antibody directed againstthe antigen is a monoclonal antibody. In some preferred embodiments,assaying the sample for antigen-antibody binding is done by precipitinreaction, radioimmunoassay, ELISA, Western blot or immunofluorescence.The invention also relates to kits for assaying bovines for a Chlamydiapsittaci infection comprising, in a suitable container: (a) apharmaceutically acceptable carrier; and (b) an antibody directedagainst a Chlamydia psittaci antigen. The method also relates to amethod of assaying for the presence of a Chlamydia psittaci infection ina bovine comprising: (a) obtaining an oligonucleotide probe comprising asequence comprised within one of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30,SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50,SEQ ID NO52:, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, or SEQ IDNO:60, or a complement thereof; and (b) employing the probe in a PCRdetection protocol. Kits for such protocols are also within the scope ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0022]FIG. 1. Scheme for Expression Library Immunization.

[0023]FIG. 2. Production of the C. psittaci Library. The C. psittacilibrary was produced by first physically shearing the genomic DNA,strain BGM/B577, and size selecting fragments of 300-800 base pairs. Thefragments were ligated into the Bgl II site of pCMVi-Ubs(+P3); see Sykesand Johnston, 1999 for details. The nucleotide sequence shown in thisfigure is given as SEQ ID NO:1.

[0024]FIG. 3. Flowchart depicting the process for deconvolution of thelibraries. Each round consists of preparation of DNA samples,vaccination of mice, challenge and determination of the relativeprotection in each group.

[0025]FIG. 4. Results of protection assays in Rounds 1, 2 and 3.Protection was scored as lung weight relative to average of thevaccinated, maximum protection, positive control and the non-vaccinated,challenged, maximum disease, negative control. The relative protectionscore was calculated by assigning the score 1 to animals with lungweight equal to the vaccinated control and the score 0 to animals withlung weights equal to the challenged, non-vaccinated control. Thesepoints define a line; animals with lower lung weight, hence betterprotection, have a higher relative protection score. Animals that haveworse disease than challenged, non-vaccinated controls, i.e. heavierlungs, will have a negative relative protection score. The unchallengedNaive group consistently had lung weights slightly lower than themaximum protection, positive controls (Vaccinated) due to theperibronchiolar accumulation of lymphatic cells. In Rounds 2 and 3 thepools of plasmids from columns in the two-dimensional arrays areassigned numbers and the rows assigned letters. The solid bars indicatepools that were designated as protective and entered into the subsequentround. The error bars represent one standard deviation on either side ofthe mean.

[0026]FIG. 5. Results of protection assays of testing individual genefragments in Round 4. Protection was scored as lung weight relative tothe average of the vaccinated, maximum protection, positive control(Vaccinated=1) and the non-vaccinated, challenged, maximum disease,negative control (Challenged=0). The Pool<50AA is the DNA consisting ofthe pool of the 32 plasmids from Round 3 having predicted open-readingframes less than 50 amino acids long. Pool>50AA is the DNA consisting ofall the 14 plasmids containing C. psittaci inserts encoding in-frameproteins more than 50 amino acids long. The numbers of each individualgene fragment tested correspond to the numbers in FIG. 4. The error barsrepresent one standard deviation of the mean.

[0027]FIG. 6. Summary of characterization of the single gene fragmentsof Round 4. The Relative Protection score of each C. psittaci (CP) genefragment is provided along with the designation of the gene in C.pneumonia that has the highest similarity (C. pneumonia homolog). In twocases, gene fragment CP #4 and CP #12, the C. psittaci gene could alsobe identified. On the right is a linear map showing the location in eachgene of the fragment that conferred protection (shaded).

[0028]FIG. 7. Protection data from DNA pools. CP1-6 is a negative poolfrom round 1. To test whether a single protective gene could be detectedin a negative pool, 25 ng of either CP4 #4 or CP4 #11 was added to 50 μgof CP1-6.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] The present invention provides compositions and methods for theimmunization of vertebrate animals, including humans, against infectionsusing nucleic acid sequences and polypeptides elucidated by screeningChlamydia psittaci. These compositions and methods will be useful forimmunization against Chlamydia psittaci bacterial infections and otherinfections and disease states. In particular embodiments, a vaccinecomposition directed against Chlamydia psittaci infections is provided.The vaccine according to the present invention comprises Chlamydiapsittaci genes and polynucleotides identified by the inventors, thatconfer protective resistance in vertebrate animals to Chlamydia psittacibacterial infections, and other infections. In other embodiments, theinvention provides methods for immunizing an animal against Chlamydiapsittaci infections, methods for preparing a cloned library viaexpression library immunization and methods for screening andidentifying Chlamydia psittaci genes that confer protection againstinfection.

[0030] A. Expression Library Immunization

[0031] In particular embodiments, the immunization of vertebrate animalsaccording to the present invention includes a cloned library ofChlamydia psittaci expression constructs. In specific embodiments, acloned expression library of Chlamydia psittaci is provided. Expressionlibrary immunization, ELI herein, is well known in the art (U.S. Pat.No. 5,703,057, specifically incorporated herein by reference). Incertain embodiments, the invention provides an ELI method applicable tovirtually any pathogen and requires no knowledge of the biologicalproperties of the pathogen. The method operates on the assumption,generally accepted by those skilled in the art, that all the potentialantigenic determinants of any pathogen are encoded in its genome. Theinventors have previously devised methods of identifying vaccines usinga genomic expression library representing all of the antigenicdeterminants of a pathogen (U.S. Pat. No. 5,703,057). The method uses toits advantage the simplicity of genetic immunization to sort through agenome for immunological reagents in an unbiased, systematic fashion.

[0032] The preparation of an expression library is performed using thetechniques and methods familiar one of skill in the art. The pathogen'sgenome, may or may not be known or possibly may even have been cloned.Thus one obtains DNA (or cDNA), representing substantially the entiregenome of the pathogen (e.g., Chlamydia psittaci). The DNA is broken up,by physical fragmentation or restriction endonuclease, into segments ofsome length so as to provide a library of about 10⁵ (approximately 18×the genome size) members. The library is then tested by inoculating asubject with purified DNA of the library or sub-library and the subjectchallenged with a pathogen, wherein immune protection of the subjectfrom pathogen challenge indicates a clone that confers a protectiveimmune response against infection.

[0033] B. Nucleic Acids

[0034] The present invention provides Chlamydia psittaci polynucleotidecompositions and methods that induce a protective immune response invertebrate animals challenged with a Chlamydia psittaci bacterialinfection. The preparation and purification of antigenic Chlamydiapsittaci polypeptides, or fragments thereof (Section C) and antibodypreparations directed against Chlamydia psittaci antigens, or fragmentsthereof (Section E) are described below.

[0035] Thus, in certain embodiments of the present invention, genes orpolynucleotides encoding Chlamydia psittaci polypeptides or fragmentsthereof are provided. It is contemplated in other embodiments, that apolynucleotide encoding a Chlamydia psittaci polypeptide or polypeptidefragment will be expressed in prokaryotic or eukaryotic cells and thepolypeptides purified for use as anti-Chlamydia psittaci antigens in thevaccination of vertebrate animals or in generating antibodiesimmunoreactive with Chlamydia psittaci polypeptides (i.e., antigens).The genomes of C. pneumoniae and C. trachomatis have been completelysequenced. The Chlamydia genes are quite similar, with the four mostprotective genes identified being 30-71% identical and 45-85% similar inamino acid sequence.

[0036] 1. Nucleic Acids Encoding Chlamydia psittaci Polypeptides

[0037] The present invention provides polynucleotides encoding antigenicChlamydia psittaci polypeptides capable of inducing a protective immuneresponse in vertebrate animals and for use as an antigen to generateanti-Chlamydia psittaci or other pathogen antibodies. In certaininstances, it may be desirable to express Chlamydia psittacipolynucleotides encoding a particular antigenic Chlamydia psittacipolypeptide domain or sequence to be used as a vaccine or in generatinganti-Chlamydia psittaci or other pathogen antibodies. Nucleic acidsaccording to the present invention may encode an entire Chlamydiapsittaci gene, or any other fragment of the Chlamydia psittaci sequencesset forth herein. The nucleic acid may be derived from genomic DNA,i.e., cloned directly from the genome of a particular organism. In otherembodiments, however, the nucleic acid may comprise complementary DNA(cDNA). A protein may be derived from the designated sequences for usein a vaccine or to isolate useful antibodies.

[0038] The term “cDNA” is intended to refer to DNA prepared usingmessenger RNA (mRNA) as template. The advantage of using a cDNA, asopposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There may be times whenthe full or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression.

[0039] It also is contemplated that a given Chlamydia psittacipolynucleotide may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the samepolypeptide (see Table 2 below). In addition, it is contemplated that agiven Chlamydia psittaci polypeptide may be generated using alternatecodons that result in a different nucleic acid sequence but encodes thesame polypeptide.

[0040] As used in this application, the term “a nucleic acid encoding aChlamydia psittaci polynucleotide” refers to a nucleic acid moleculethat has been isolated free of total cellular nucleic acid. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine (Table 2, below), and also refers to codons that encodebiologically equivalent amino acids, as discussed in the followingpages. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

[0041] Allowing for the degeneracy of the genetic code, sequences thathave at least about 50%, usually at least about 60%, more usually about70%, most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of given Chlamydia psittaci gene or polynucleotide.Sequences that are essentially the same as those set forth in aChlamydia psittaci gene or polynucleotide may also be functionallydefined as sequences that are capable of hybridizing to a nucleic acidsegment containing the complement of a Chlamydia psittaci polynucleotideunder standard conditions.

[0042] The DNA segments of the present invention include those encodingfunctional and/or immunologically equivalent Chlamydia psittaci proteinsand peptides, as described above. Such sequences may arise as aconsequence of codon redundancy and amino acid functional equivalencythat are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally and/orimmunogenically equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced through the application of site-directed mutagenesistechniques or may be introduced randomly and screened later for thedesired function, as described below.

[0043] 2. Oligonucleotide Sequences

[0044] Naturally, the present invention also encompasses DNA segmentsthat are complementary, or essentially complementary to the sequences ofa Chlamydia psittaci polynucleotide. Nucleic acid sequences that are“complementary” are those that are capable of base-pairing according tothe standard Watson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of a Chlamydia psittacipolynucleotide under relatively stringent conditions such as thosedescribed herein. Such sequences may encode the entire Chlamydiapsittaci polypeptide or functional or non-functional fragments thereof.

[0045] Alternatively, the hybridizing segments may be shorteroligonucleotides. Sequences of 17 bases long should occur only once inthe human genome and, therefore, suffice to specify a unique targetsequence. Although shorter oligomers are easier to make and increase invivo accessibility, numerous other factors are involved in determiningthe specificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or3500 bases and longer are contemplated as well. Such oligonucleotideswill find use, for example, as probes in Southern and Northern blots andas primers in amplification reactions, or for vaccines.

[0046] Suitable hybridization conditions will be well known to those ofskill in the art. In certain applications, for example, substitution ofamino acids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

[0047] In other embodiments, hybridization may be achieved underconditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 MMMgCl₂, 10 mM dithiothreitol, at temperatures between approximately 20°C. to about 37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

[0048] One method of using probes and primers of the present inventionis in the search for genes related to Chlamydia psittaci or, moreparticularly, homologs of Chlamydia psittaci from other species.Normally, the target DNA will be a genomic or cDNA library, althoughscreening may involve analysis of RNA molecules. By varying thestringency of hybridization, and the region of the probe, differentdegrees of homology may be discovered.

[0049] Another way of exploiting probes and primers of the presentinvention is in site-directed, or site-specific mutagenesis.Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

[0050] The technique typically employs a bacteriophage vector thatexists in both a single stranded and double stranded form. Typicalvectors useful in site-directed mutagenesis include vectors such as theM13 phage. These phage vectors are commercially available and their useis generally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

[0051] In general, site-directed mutagenesis is performed by firstobtaining a single-stranded vector, or melting of two strands of adouble stranded vector which includes within its sequence a DNA sequenceencoding the desired protein. An oligonucleotide primer bearing thedesired mutated sequence is synthetically prepared. This primer is thenannealed with the single-stranded DNA preparation, taking into accountthe degree of mismatch when selecting hybridization conditions, andsubjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected that include recombinant vectors bearing the mutated sequencearrangement.

[0052] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

[0053] C. Polypeptides and Antigens

[0054] For the purposes of the present invention a Chlamydia psittacipolypeptide used as an antigen may be a naturally-occurring Chlamydiapsittaci polypeptide that has been extracted using protein extractiontechniques well known to those of skill in the art. In particularembodiments, a Chlamydia psittaci antigen is identified by ELI andprepared in a pharmaceutically acceptable carrier for the vaccination ofan animal against Chlamydia psittaci infection.

[0055] In alternative embodiments, the Chlamydia psittaci polypeptide orantigen may be a synthetic peptide. In still other embodiments, thepeptide may be a recombinant peptide produced through molecularengineering techniques. The present section describes the methods andcompositions involved in producing a composition of Chlamydia psittacipolypeptides for use as antigens in the present invention.

[0056] 1. Chlamydia psittaci Polypeptides as Antigens

[0057] Section A describes methods for preparing a cloned Chlamydiapsittaci library for ELI. Described also are methods for screening andidentifying Chlamydia psittaci genes that confer protection againstChlamydia psittaci infection. Thus, Chlamydia psittaci polypeptideencoding genes or their corresponding cDNA identified in the presentinvention can be inserted into an appropriate cloning vehicle for theproduction of Chlamydia psittaci polypeptides as antigens for thepresent invention. In addition, sequence variants of the polypeptide canbe prepared. These may, for instance, be minor sequence variants of thepolypeptide that arise due to natural variation within the population orthey may be homologues found in other species. They also may besequences that do not occur naturally, but that are sufficiently similarthat they function similarly and/or elicit an immune response thatcross-reacts with natural forms of the polypeptide. Sequence variantscan be prepared by standard methods of site-directed mutagenesis such asthose described below in the following section.

[0058] Another synthetic or recombinant variation of a Chlamydiapsittaci-antigen is a polyepitopic moiety comprising repeats of epitopicdeterminants found naturally on Chlamydia psittaci proteins. Suchsynthetic polyepitopic proteins can be made up of several homomericrepeats of any one Chlamydia psittaci protein epitope; or can compriseof two or more heteromeric epitopes expressed on one or severalChlamydia psittaci protein epitopes.

[0059] Amino acid sequence variants of the polypeptide can besubstitutional, insertional or deletion variants. Deletion variants lackone or more residues of the native protein which are not essential forfunction or immunogenic activity, and are exemplified by the variantslacking a transmembrane sequence described above. Another common type ofdeletion variant is one lacking secretory signal sequences or signalsequences directing a protein to bind to a particular part of a cell.

[0060] Substitutional variants typically contain the exchange of oneamino acid for another at one or more sites within the protein, and maybe designed to modulate one or more properties of the polypeptide suchas stability against proteolytic cleavage. Substitutions preferably areconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

[0061] Insertional variants include fusion proteins such as those usedto allow rapid purification of the polypeptide and also can includehybrid proteins containing sequences from other proteins andpolypeptides which are homologues of the polypeptide. For example, aninsertional variant could include portions of the amino acid sequence ofthe polypeptide from one species, together with portions of thehomologous polypeptide from another species. Other insertional variantscan include those in which additional amino acids are introduced withinthe coding sequence of the polypeptide. These typically are smallerinsertions than the fusion proteins described above and are introduced,for example, into a protease cleavage site.

[0062] In one embodiment, major antigenic determinants of thepolypeptide may be identified by an empirical approach in which portionsof the gene encoding the polypeptide are expressed in a recombinanthost, and the resulting proteins tested for their ability to elicit animmune response. For example, the polymerase chain reaction (PCR) can beused to prepare a range of cDNAs encoding peptides lacking successivelylonger fragments of the C-terminus of the protein. The immunogenicactivity of each of these peptides then identifies those fragments ordomains of the polypeptide that are essential for this activity. Furtherexperiments in which only a small number of amino acids are removed oradded at each iteration then allows the location of other antigenicdeterminants of the polypeptide. Thus, the polymerase chain reaction, atechnique for amplifying a specific segment of DNA via multiple cyclesof denaturation-renaturation, using a thermostable DNA polymerase,deoxyribonucleotides and primer sequences is contemplated in the presentinvention (Mullis, 1990; Mullis et al., 1992).

[0063] Another embodiment for the preparation of the polypeptidesaccording to the invention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. Because many proteins exert their biological activity viarelatively small regions of their folded surfaces, their actions can bereproduced by much smaller designer (mimetic) molecules that retain thebioactive surfaces and have potentially improved pharmacokinetic/dynamicproperties (Fairlie et al., 1998).

[0064] The underlying rationale behind the use of peptide mimetics isthat the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. However, unlike proteins,peptides often lack well defined three dimensional structure in aqueoussolution and tend to be conformationally mobile. Progress has been madewith the use of molecular constraints to stabilize the bioactiveconformations. By affixing or incorporating templates that fix secondaryand tertiary structures of small peptides, synthetic molecules (proteinsurface mimetics) can be devised to mimic the localized elements ofprotein structure that constitute bioactive surfaces. Methods formimicking individual elements of secondary structure (helices, turns,strands, sheets) and for assembling their combinations into tertiarystructures (helix bundles, multiple loops, helix-loop-helix motifs) havebeen reviewed (Fairlie et al., 1998; Moore, 1994).

[0065] Methods for predicting, preparing, modifying, and screeningmimetic peptides are described in U.S. Pat. No. 5,933,819 and U.S. Pat.No. 5,869,451 (each specifically incorporated herein by reference). Itis contemplated in the present invention, that peptide mimetics will beuseful in screening modulators of an immune response.

[0066] Modifications and changes may be made in the structure of a geneand still obtain a functional molecule that encodes a protein orpolypeptide with desirable characteristics. The following is adiscussion based upon changing the amino acids of a protein to create anequivalent, or even an improved, second-generation molecule. The aminoacid changes may be achieved by changing the codons of the DNA sequence,according to the following data.

[0067] For example, certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid substitutions can be made in a protein sequence, and itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventor that variouschanges may be made in the DNA sequences of genes without appreciableloss of their biological utility or activity. Table 1 shows the codonsthat encode particular amino acids.

[0068] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982).

[0069] It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0070] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics (Kyte &Doolittle, 1982), these are: Isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0071] It is known in the art that certain amino acids may besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biological functionally equivalent protein. Inmaking such changes, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

[0072] It also is understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

[0073] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine *−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0074] It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent and immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those that are within +1 are particularlypreferred, and those within +0.5 are even more particularly preferred.

[0075] As outlined above, amino acid substitutions generally are basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

[0076] 2. Synthetic Polypeptides

[0077] Contemplated in the present invention are Chlamydia psittaciproteins and related peptides for use as antigens. In certainembodiments, the synthesis of a Chlamydia psittaci peptide fragment isconsidered. The peptides of the invention can be synthesized in solutionor on a solid support in accordance with conventional techniques.Various automatic synthesizers are commercially available and can beused in accordance with known protocols. See, for example, Stewart andYoung, (1984); Tam et al., (1983); Merrifield, (1986); and Barany andMerrifield (1979), each incorporated herein by reference. Alternatively,recombinant DNA technology may be employed wherein a nucleotide sequencewhich encodes a peptide of the invention is inserted into an expressionvector, transformed or transfected into an appropriate host cell andcultivated under conditions suitable for expression.

[0078] 3. Chlamydia psittaci Polypeptide/Antigen Purification

[0079]Chlamydia psittaci polypeptides of the present invention are usedas antigens for inducing a protective immune response in an animal andfor the preparation of anti-Chlamydia psittaci antibodies. Thus, certainaspects of the present invention concern the purification, and inparticular embodiments, the substantial purification, of a Chlamydiapsittaci polypeptide that is described herein above. The term “purifiedprotein or peptide” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the protein orpeptide is purified to any degree relative to its naturally-obtainablestate. A purified protein or peptide therefore also refers to a proteinor peptide, free from the environment in which it may naturally occur.

[0080] Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50% or more of the proteins in the composition.

[0081] Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the number ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

[0082] Various techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

[0083] There is no general requirement that the protein or peptidealways be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater-foldpurification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

[0084] It is known that the migration of a polypeptide can vary,sometimes significantly, with different conditions of SDS/PAGE (Capaldiet al., 1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

[0085] High Performance Liquid Chromatography (HPLC) is characterized bya very rapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainand adequate flow rate. Separation can be accomplished in a matter ofminutes, or a most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

[0086] Gel chromatography, or molecular sieve chromatography, is aspecial type of partition chromatography that is based on molecularsize. The theory behind gel chromatography is that the column, which isprepared with tiny particles of an inert substance that contain smallpores, separates larger molecules from smaller molecules as they passthrough or around the pores, depending on their size. As long as thematerial of which the particles are made does not adsorb the molecules,the sole factor determining rate of flow is the size. Hence, moleculesare eluted from the column in decreasing size, so long as the shape isrelatively constant. Gel chromatography is unsurpassed for separatingmolecules of different size because separation is independent of allother factors such as pH, ionic strength, temperature, etc. There alsois virtually no adsorption, less zone spreading and the elution volumeis related in a simple matter to molecular weight.

[0087] Affinity Chromatography is a chromatographic procedure thatrelies on the specific affinity between a substance to be isolated and amolecule that it can specifically bind to. This is a receptor-ligandtype interaction. The column material is synthesized by covalentlycoupling one of the binding partners to an insoluble matrix. The columnmaterial is then able to specifically adsorb the substance from thesolution. Elution occurs by changing the conditions to those in whichbinding will not occur (alter pH, ionic strength, temperature, etc.).

[0088] D. Gene Delivery

[0089] In certain embodiments of the invention, an expression constructcomprising a Chlamydia psittaci gene or polynucleotide segment under thecontrol of a heterologous promoter operable in eukaryotic cells isprovided. The general approach in certain aspects of the presentinvention is to provide a cell with an expression construct encoding aspecific Chlamydia psittaci protein, polypeptide or peptide fragment,thereby permitting the antigenic expression of the protein, polypeptideor peptide fragment to take effect in the cell. Following delivery ofthe expression construct, the protein, polypeptide or peptide fragmentencoded by the expression construct is synthesized by thetranscriptional and translational machinery of the cell, as well as anythat may be provided by the expression construct.

[0090] Viral and non-viral vector systems are the two predominatecategories for the delivery of an expression construct encoding atherapeutic protein, polypeptide, polypeptide fragment. Both vectorsystems are described in the following sections. There also are twoprimary approaches utilized in the delivery of an expression constructfor the purposes of gene therapy; either indirect, ex vivo methods ordirect, in vivo methods. Ex vivo gene transfer comprises vectormodification of (host) cells in culture and the administration ortransplantation of the vector modified cells to a gene therapyrecipient. In vivo gene transfer comprises direct introduction of thevector (e.g., injection, inhalation) into the target source ortherapeutic gene recipient.

[0091] In certain embodiments of the invention, the nucleic acidencoding the gene or polynucleotide may be stably integrated into thegenome of the cell. In yet further embodiments, the nucleic acid may bestably or transiently maintained in the cell as a separate, episomalsegment of DNA. Such nucleic acid segments or “episomes” encodesequences sufficient to permit maintenance and replication independentof or in synchronization with the host cell cycle. How the expressionconstruct is delivered to a cell and/or where in the cell the nucleicacid remains is dependent on the type of vector employed. The followinggene delivery methods provide the framework for choosing and developingthe most appropriate gene delivery system for a preferred application.

[0092] 1. Non-Viral Polynucleotide Delivery

[0093] In one embodiment of the invention, a polynucleotide expressionconstruct consists of naked recombinant DNA or plasmids. In preferredembodiments of the invention, a Chlamydia psittaci polynucleotide isadministered to a subject via injection and/or particle bombardment(e.g., a gene gun). Thus, in one preferred embodiment, polynucleotideexpression constructs are transferred into cells by acceleratingDNA-coated microprojectiles to a high velocity, allowing the DNA-coatedmicroprojectiles to pierce cell membranes and enter cells. In anotherpreferred embodiment, polynucleotides are administered to a subject byinjection. Injection of a polynucleotide expression construct may begiven by intramuscular, intravenous, subcutaneous, ir intraperitonealinjection, as long as the polynucleotide expression construct caneffectively be delivered to a desired target.

[0094] a. Particle Bombardment

[0095] Particle Bombardment depends on the ability to accelerateDNA-coated microprojectiles to a high velocity allowing them to piercecell membranes and enter cells without killing them (Klein et al.,1987). Several devices for accelerating small particles have beendeveloped. The most commonly used forms rely on high-pressure helium gas(Sanford et al., 1991), of which one of the present inventors is aco-inventor. The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

[0096] For microprojectile bombardment transformation using theconstructs of the instant invention, both physical and biologicalparameters may be optimized. Physical factors are those that involvemanipulating the DNA/microprojectile precipitate or those that affectthe flight and velocity of either the macro- or microprojectiles.Biological factors include all steps involved in manipulation of cellsbefore and immediately after bombardment, such as the osmotic adjustmentof target cells to help alleviate the trauma associated withbombardment, the orientation of an immature embryo or other targettissue relative to the particle trajectory, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.

[0097] Accordingly, it is contemplated that one may wish to adjustvarious bombardment parameters in small scale studies to fully optimizethe conditions. One may particularly wish to adjust physical parameterssuch as DNA concentration, gap distance, flight distance, tissuedistance, and helium pressure. It is further contemplated that the gradeof helium may effect transformation efficiency. One also may optimizethe trauma reduction factors (TRFs) by modifying conditions whichinfluence the physiological state of the recipient cells and which maytherefore influence transformation and integration efficiencies. Forexample, the osmotic state, tissue hydration and the subculture stage orcell cycle of the recipient cells may be adjusted for optimumtransformation.

[0098] Other physical factors include those that involve manipulatingthe DNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells immediately beforeand after bombardment. The pre-bombardment culturing conditions, such asosmotic environment, the bombardment parameters, and the plasmidconfiguration have been adjusted to yield the maximum numbers of stabletransformants.

[0099] For microprojectile bombardment, one will attach (i.e., “coat”)DNA to the microprojectiles such that it is delivered to recipient cellsin a form suitable for transformation thereof. In this respect, at leastsome of the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of interactions between transforming DNA and themicroprojectile following delivery of the microprojectile to the targetcell. This need not be the case, however, as availability to a targetcell may occur as a result of breakage of unbound segments of DNA or ofother molecules which comprise the physical attachment to themicroprojectile. Availability may further occur as a result of breakageof bonds between the transforming DNA and other molecules, which areeither directly or indirectly attached to the microprojectile. It isfurther contemplated that transformation of a target cell may occur byway of direct illegitimate or homology-dependent recombination betweenthe transforming DNA and the genomic DNA of the recipient cell.Therefore, as used herein, a “coated” microprojectile will be one whichis capable of being used to transform a target cell, in that thetransforming DNA will be delivered to the target cell, yet will beaccessible to the target cell such that transformation may occur.

[0100] Any technique for coating microprojectiles which allows fordelivery of transforming DNA to the target cells may be used. Methodsfor coating microprojectiles which have been demonstrated to work wellwith the current invention have been specifically disclosed herein. DNAmay be bound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend labeled with long chain thiol cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction, but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

[0101] Alternatively, particles may be prepared by functionalizing thesurface of a gold oxide particle, providing free amine groups. DNA,having a strong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

[0102] b. Other Non-Viral Methods of Polynucleotide Delivery

[0103] Transfer of a cloned expression construct in the presentinvention also may be performed by any of the methods which physicallyor chemically permeabilize the cell membrane (e.g., calcium phosphateprecipitation, DEAE-dextran, electroporation, direct microinjection,DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication,gene bombardment using high velocity microprojectiles andreceptor-mediated transfection.

[0104] In certain embodiments, the use of lipid formulations and/ornanocapsules is contemplated for the introduction of a Chlamydiapsittaci polynucleotide or polypeptide, or a gene therapy vector intohost cells.

[0105] Nanocapsules can generally entrap compounds in a stable and/orreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and/or such particles maybe easily made.

[0106] In a preferred embodiment of the invention, the polynucleotide orpolypeptide may be associated with a lipid. The polynucleotide orpolypeptide associated with a lipid may be encapsulated in the aqueousinterior of a liposome, interspersed within the lipid bilayer of aliposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the oligonucleotide, entrapped ina liposome, complexed with a liposome, dispersed in a solutioncontaining a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, orotherwise associated with a lipid. The lipid or lipid/polynucleotide orpolypeptide associated compositions of the present invention are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates which are not uniform in either size or shape.

[0107] Lipids suitable for use according to the present invention can beobtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) can be obtained from Sigma Chemical Co.,dicetyl phosphate (“DCP”) is obtained from K & K Laboratories(Plainview, N.Y.); cholesterol (“Chol”) is obtained fromCalbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and otherlipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Preferably, chloroform is used as theonly solvent since it is more readily evaporated than methanol.

[0108] “Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes may be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). However, the present invention also encompassescompositions that have different structures in solution than the normalvesicular structure. For example, the lipids may assume a micellarstructure or merely exist as non-uniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

[0109] Liposomes within the scope of the present invention can beprepared in accordance with known laboratory procedures, for example:the method of Bangham et al. (1965), the contents of which areincorporated herein by reference; the method of Gregoriadis, asdescribed in DRUG CARRIERS IN BIOLOGY AND MEDICINE, G. Gregoriadis ed.(1979) pp. 287-341, the contents of which are incorporated herein byreference; the method of Deamer and Uster (1983), the contents of whichare incorporated by reference; and the reverse-phase evaporation methodas described by Szoka and Papahadjopoulos (1978).

[0110] Other vector delivery systems which can be employed to deliver anucleic acid encoding a therapeutic gene into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific(Wu and Wu, 1993).

[0111] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

[0112] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al. (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding atherapeutic gene also may be specifically delivered into a cell typesuch as prostate, epithelial or tumor endothelial cells, by any numberof receptor-ligand systems with or without liposomes. For example, thehuman prostate-specific antigen (Watt et al., 1986) may be used as thereceptor for mediated delivery of a nucleic acid in prostate tissue.

[0113] In another embodiment of the invention, the expression constructmay simply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically pertneabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. (1984) successfullyinjected polyomavirus DNA in the form of CaPO₄ precipitates into liverand spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of CaPO₄ precipitatedplasmids results in expression of the transfected genes. It isenvisioned that DNA encoding a Chlamydia psittaci gene or polynucleotideof interest may also be transferred in a similar manner in vivo andexpress the gene or polynucleotide product.

[0114] 2. Viral Vectors

[0115] In certain embodiments, it is contemplated that a Chlamydiapsittaci gene or polynucleotide that confers immune resistance toinfection may be delivered by a viral vector. The capacity of certainviral vectors to efficiently infect or enter cells, to integrate into ahost cell genome and stably express viral genes, have led to thedevelopment and application of a number of different viral vectorsystems (Robbins et al., 1998). Viral systems are currently beingdeveloped for use as vectors for ex vivo and in vivo gene transfer. Forexample, adenovirus, herpes-simple virus, retrovirus andadeno-associated virus vectors are being evaluated currently fortreatment of diseases such as cancer, cystic fibrosis, Gaucher disease,renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al.,1998; U.S. Pat. No. 5,670,488). The various viral vectors describedbelow, present specific advantages and disadvantages, depending on theparticular gene-therapeutic application.

[0116] a. Adenoviral Vectors

[0117] In particular embodiments, an adenoviral expression vector iscontemplated for the delivery of expression constructs. “Adenovirusexpression vector” is meant to include those constructs containingadenovirus sequences sufficient to (a) support packaging of theconstruct and (b) to ultimately express a tissue or cell-specificconstruct that has been cloned therein.

[0118] Adenoviruses comprise linear double stranded DNA, with a genomeranging from 30 to 35 kb in size (Reddy et al., 1998; Morrison et al.,1997; Chillon et al., 1999). An adenovirus expression vector accordingto the present invention comprises a genetically engineered form of theadenovirus. Advantages of adenoviral gene transfer include the abilityto infect a wide variety of cell types, including non-dividing cells, amid-sized genome, ease of manipulation, high infectivity and they can begrown to high titers (Wilson, 1996). Further, adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner, without potential genotoxicityassociated with other viral vectors. Adenoviruses also are structurallystable (Marienfeld et al., 1999) and no genome rearrangement has beendetected after extensive amplification (Parks et al., 1997; Bett et al.,1993).

[0119] Salient features of the adenovirus genome are an early region(E1, E2, E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene),a late region (L1, L2, L3, L4 and L5 genes), a major late promoter(MLP), inverted-terminal-repeats (ITRs) and a ψ sequence (Zheng, et al.,1999; Robbins et al., 1998; Graham and Prevec, 1995). The early genesE1, E2, E3 and E4 are expressed from the virus after infection andencode polypeptides that regulate viral gene expression, cellular geneexpression, viral replication, and inhibition of cellular apoptosis.Further on during viral infection, the MLP is activated, resulting inthe expression of the late (L) genes, encoding polypeptides required foradenovirus encapsidation. The intermediate region encodes components ofthe adenoviral capsid. Adenoviral inverted terminal repeats (ITRs;100-200 bp in length), are cis elements, function as origins ofreplication and are necessary for viral DNA replication. The ψ sequenceis required for the packaging of the adenoviral genome.

[0120] A common approach for generating an adenoviruses for use as agene transfer vector is the deletion of the E1 gene (E1⁻), which isinvolved in the induction of the E2, E3 and E4 promoters (Graham andPrevec, 1995). Subsequently, a therapeutic gene or genes can be insertedrecombinantly in place of the E1 gene, wherein expression of thetherapeutic gene(s) is driven by the E1 promoter or a heterologouspromoter. The E1⁻, replication-deficient virus is then proliferated in a“helper” cell line that provides the E1 polypeptides in trans (e.g., thehuman embryonic kidney cell line 293). Thus, in the present invention itmay be convenient to introduce the transforming construct at theposition from which the E1-coding sequences have been removed. However,the position of insertion of the construct within the adenovirussequences is not critical to the invention. Alternatively, the E3region, portions of the E4 region or both may be deleted, wherein aheterologous nucleic acid sequence under the control of a promoteroperable in eukaryotic cells is inserted into the adenovirus genome foruse in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210,each specifically incorporated herein by reference).

[0121] Although adenovirus based vectors offer several unique advantagesover other vector systems, they often are limited by vectorimmunogenicity, size constraints for insertion of recombinant genes andlow levels of replication. The preparation of a recombinant adenovirusvector deleted of all open reading frames, comprising a full lengthdystrophin gene and the terminal repeats required for replication(Haecker et al., 1997) offers some potentially promising advantages tothe above mentioned adenoviral shortcomings. The vector was grown tohigh titer with a helper virus in 293 cells and was capable ofefficiently transducing dystrophin in mdx mice, in myotubes in vitro andmuscle fibers in vivo. Helper-dependent viral vectors are discussedbelow.

[0122] A major concern in using adenoviral vectors is the generation ofa replication-competent virus during vector production in a packagingcell line or during gene therapy treatment of an individual. Thegeneration of a replication-competent virus could pose serious threat ofan unintended viral infection and pathological consequences for thepatient. Armentano et al., describe the preparation of areplication-defective adenovirus vector, claimed to eliminate thepotential for the inadvertent generation of a replication-competentadenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein byreference). The replication-defective adenovirus method comprises adeleted El region and a relocated protein 1×gene, wherein the vectorexpresses a heterologous, mammalian gene.

[0123] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes and/or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0124] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus Elregion. Adenovirus growth and manipulation is known to those of skill inthe art, and exhibits broad host range in vitro and in vivo (U.S. Pat.No. 5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,54). Thisgroup of viruses can be obtained in high titers, e.g., 10⁹ to 10¹¹plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. Many experiments,innovations, preclinical studies and clinical trials are currently underinvestigation for the use of adenoviruses as gene delivery vectors. Forexample, adenoviral gene delivery-based gene therapies are beingdeveloped for liver diseases (Han et al., 1999), psychiatric diseases(Lesch, 1999), neurological diseases (Smith, 1998; Hermens andVerhaagen, 1998), coronary diseases (Feldman et al., 1996), musculardiseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) andvarious cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai etal., 1999), pancreatic (Carrion et al., 1999), bladder (Irie et al.,1999), head and neck (Blackwell et al., 1999), breast (Stewart et al.,1999), lung (Batra et al., 1999) and ovarian (Vanderkwaak et al., 1999).

[0125] b. Retroviral Vectors

[0126] In certain embodiments of the invention, the use of retrovirusesfor gene delivery are contemplated. Retroviruses are RNA virusescomprising an RNA genome. When a host cell is infected by a retrovirus,the genomic RNA is reverse transcribed into a DNA intermediate which isintegrated into the chromosomal DNA of infected cells. This integratedDNA intermediate is referred to as a provirus. A particular advantage ofretroviruses is that they can stably infect dividing cells with a geneof interest (e.g., a therapeutic gene) by integrating into the host DNA,without expressing immunogenic viral proteins. Theoretically, theintegrated retroviral vector will be maintained for the life of theinfected host cell, expressing the gene of interest.

[0127] The retroviral genome and the proviral DNA have three genes: gag,pol, and env, which are flanked by two long terminal repeat (LTR)sequences. The gag gene encodes the internal structural (matrix, capsid,and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase) and the env gene encodes viralenvelope glycoproteins. The 5′ and 3′ LTRs serve to promotetranscription and polyadenylation of the virion RNAs. The LTR containsall other cis-acting sequences necessary for viral replication.

[0128] A recombinant retrovirus of the present invention may begenetically modified in such a way that some of the structural,infectious genes of the native virus have been removed and replacedinstead with a nucleic acid sequence to be delivered to a target cell(U.S. Pat. No. 5,858,744; U.S. Pat. No. 5,739,018, each incorporatedherein by reference). After infection of a cell by the virus, the virusinjects its nucleic acid into the cell and the retrovirus geneticmaterial can integrate into the host cell genome. The transferredretrovirus genetic material is then transcribed and translated intoproteins within the host cell. As with other viral vector systems, thegeneration of a replication-competent retrovirus during vectorproduction or during therapy is a major concern. Retroviral vectorssuitable for use in the present invention are generally defectiveretroviral vectors that are capable of infecting the target cell,reverse transcribing their RNA genomes, and integrating the reversetranscribed DNA into the target cell genome, but are incapable ofreplicating within the target cell to produce infectious retroviralparticles (e.g., the retroviral genome transferred into the target cellis defective in gag, the gene encoding virion structural proteins,and/or in pol, the gene encoding reverse transcriptase). Thus,transcription of the provirus and assembly into infectious virus occursin the presence of an appropriate helper virus or in a cell linecontaining appropriate sequences enabling encapsidation withoutcoincident production of a contaminating helper virus.

[0129] The growth and maintenance of retroviruses is known in the art(U.S. Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specificallyincorporated herein by reference). Nolan et al. describe the productionof stable high titre, helper-free retrovirus comprising a heterologousgene (U.S. Pat. No. 5,830,725, specifically incorporated herein byreference). Methods for constructing packaging cell lines useful for thegeneration of helper-free recombinant retroviruses with amphoteric orecotrophic host ranges, as well as methods of using the recombinantretroviruses to introduce a gene of interest into eukaryotic cells invivo and in vitro are contemplated in the present invention (U.S. Pat.No. 5,955,331).

[0130] Currently, the majority of all clinical trials for vectormediated gene delivery use murine leukemia virus (MLV)-based retroviralvector gene delivery (Robbins et al., 1998; Miller et al., 1993).Disadvantages of retroviral gene delivery includes a requirement forongoing cell division for stable infection and a coding capacity thatprevents the delivery of large genes. However, recent development ofvectors such as lentivirus (e.g., HIV), simian immunodeficiency virus(SIV) and equine infectious-anemia virus (EIAV), which can infectcertain non-dividing cells, potentially allow the in vivo use ofretroviral vectors for gene therapy applications (Amado and Chen, 1999;Klimatcheva et al., 1999; White et al., 1999; Case et al., 1999). Forexample, HIV-based vectors have been used to infect non-dividing cellssuch as neurons (Takashi et al., 1999; Miyake et al., 1999), islets(Leibowitz et al., 1999) and muscle cells (Johnston et al., 1999). Thetherapeutic delivery of genes via retroviruses are currently beingassessed for the treatment of various disorders such as inflammatorydisease (Moldawer et al., 1999),

[0131] AIDS (Amado et al., 1999; Engel and Kohn, 1999), cancer (Clay etal., 1999), cerebrovascular disease (Weihl et al., 1999) and hemophilia(Kay, 1998).

[0132] C. Herpes-Simplex Viral Vectors

[0133] Herpes simplex virus (HSV) type I and type II contain adouble-stranded, linear DNA genome of approximately 150 kb, encoding70-80 genes. Wild type HSV are able to infect cells lytically and toestablish latency in certain cell types (e.g., neurons).

[0134] Similar to adenovirus, HSV also can infect a variety of celltypes including muscle (Yeung et al., 1999), ear (Derby et al., 1999),eye (Kaufman et al., 1999), tumors (Yoon et al., 1999; Howard et al.,1999), lung (Kohut et al., 1998), neuronal (Gamido et al., 1999;Lachmann and Efstathiou, 1999), liver (Miytake et al., 1999; Kooby etal., 1999) and pancreatic islets (Rabinovitch et al., 1999).

[0135] HSV viral genes are transcribed by cellular RNA polymerase II andare temporally regulated, resulting in the transcription and subsequentsynthesis of gene products in roughly three discernable phases orkinetic classes. These phases of genes are referred to as the ImmediateEarly (IE) or alpha genes, Early (E) or beta genes and Late (L) or gammagenes. Immediately following the arrival of the genome of a virus in thenucleus of a newly infected cell, the IE genes are transcribed. Theefficient expression of these genes does not require prior viral proteinsynthesis. The products of IE genes are required to activatetranscription and regulate the remainder of the viral genome.

[0136] For use in therapeutic gene delivery, HSV must be renderedreplication-defective. Protocols for generating replication-defectiveHSV helper virus-free cell lines have been described (U.S. Pat. No.5,879,934; U.S. Pat. No. 5,851,826, each specifically incorporatedherein by reference in its entirety). One IE protein, Infected CellPolypeptide 4 (ICP4), also known as alpha 4 or Vmw175, is absolutelyrequired for both virus infectivity and the transition from IE to latertranscription. Thus, due to its complex, multifunctional nature andcentral role in the regulation of HSV gene expression, ICP4 hastypically been the target of HSV genetic studies.

[0137] Phenotypic studies of HSV viruses deleted of ICP4 indicate thatsuch viruses will be potentially useful for gene transfer purposes(Krisky et al., 1998a). One property of viruses deleted for ICP4 thatmakes them desirable for gene transfer is that they only express thefive other IE genes: ICP0, ICP6, ICP27, ICP22 and ICP47 (DeLuca et al.,1985), without the expression of viral genes encoding proteins thatdirect viral DNA synthesis, as well as the structural proteins of thevirus. This property is desirable for minimizing possible deleteriouseffects on host cell metabolism or an immune response following genetransfer. Further deletion of IE genes ICP22 and ICP27, in addition toICP4, substantially improve reduction of HSV cytotoxicity and preventedearly and late viral gene expression (Krisky et al., 1998b).

[0138] The therapeutic potential of HSV in gene transfer has beendemonstrated in various in vitro model systems and in vivo for diseasessuch as Parkinson's (Yamada et al., 1999), retinoblastoma (Hayashi etal., 1999), intracerebral and intradermal tumors (Moriuchi et al.,1998), B cell malignancies (Suzuki et al., 1998), ovarian cancer (Wanget al., 1998) and Duchenne muscular dystrophy (Huard et al., 1997).

[0139] d. Adeno-Associated Viral Vectors

[0140] Adeno-associated virus (AAV), a member of the parvovirus family,is a human virus that is increasingly being used for gene deliverytherapeutics. AAV has several advantageous features not found in otherviral systems. First, AAV can infect a wide range of host cells,including non-dividing cells. Second, AAV can infect cells fromdifferent species. Third, AAV has not been associated with any human oranimal disease and does not appear to alter the biological properties ofthe host cell upon integration. For example, it is estimated that 80-85%of the human population has been exposed to AAV. Finally, AAV is stableat a wide range of physical and chemical conditions which lends itselfto production, storage and transportation requirements.

[0141] The AAV genome is a linear, single-stranded DNA moleculecontaining 4681 nucleotides. The AAV genome generally comprises aninternal non-repeating genome flanked on each end by inverted terminalrepeats (ITRs) of approximately 145 bp in length. The ITRs have multiplefunctions, including origins of DNA replication, and as packagingsignals for the viral genome. The internal non-repeated portion of thegenome includes two large open reading frames, known as the AAVreplication (rep) and capsid (cap) genes. The rep and cap genes code forviral proteins that allow the virus to replicate and package the viralgenome into a virion. A family of at least four viral proteins areexpressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40,named according to their apparent molecular weight. The AAV cap regionencodes at least three proteins, VP1, VP2, and VP3.

[0142] AAV is a helper-dependent virus requiring co-infection with ahelper virus (e.g., adenovirus, herpesvirus or vaccinia) in order toform AAV virions. In the absence of co-infection with a helper virus,AAV establishes a latent state in which the viral genome inserts into ahost cell chromosome, but infectious virions are not produced.Subsequent infection by a helper virus “rescues” the integrated genome,allowing it to replicate and package its genome into infectious AAVvirions. Although AAV can infect cells from different species, thehelper virus must be of the same species as the host cell (e.g., humanAAV will replicate in canine cells co-infected with a canineadenovirus).

[0143] AAV has been engineered to deliver genes of interest by deletingthe internal non-repeating portion of the AAV genome and inserting aheterologous gene between the ITRs. The heterologous gene may befunctionally linked to a heterologous promoter (constitutive,cell-specific, or inducible) capable of driving gene expression intarget cells. To produce infectious recombinant AAV (rAAV) containing aheterologous gene, a suitable producer cell line is transfected with arAAV vector containing a heterologous gene. The producer cell isconcurrently transfected with a second plasmid harboring the AAV rep andcap genes under the control of their respective endogenous promoters orheterologous promoters. Finally, the producer cell is infected with ahelper virus.

[0144] Once these factors come together, the heterologous gene isreplicated and packaged as though it were a wild-type AAV genome. Whentarget cells are infected with the resulting rAAV virions, theheterologous gene enters and is expressed in the target cells. Becausethe target cells lack the rep and cap genes and the adenovirus helpergenes, the rAAV cannot further replicate, package or form wild-type AAV.

[0145] The use of helper virus, however, presents a number of problems.First, the use of adenovirus in a rAAV production system causes the hostcells to produce both rAAV and infectious adenovirus. The contaminatinginfectious adenovirus can be inactivated by heat treatment (56.degree.C. for 1 hour). Heat treatment, however, results in approximately a 50%drop in the titer of functional rAAV virions. Second, varying amounts ofadenovirus proteins are present in these preparations. For example,approximately 50% or greater of the total protein obtained in such rAAVvirion preparations is free adenovirus fiber protein. If not completelyremoved, these adenovirus proteins have the potential of eliciting animmune response from the patient. Third, AAV vector production methodswhich employ a helper virus require the use and manipulation of largeamounts of high titer infectious helper virus, which presents a numberof health and safety concerns, particularly in regard to the use of aherpesvirus. Fourth, concomitant production of helper virus particles inrAAV virion producing cells diverts large amounts of host cellularresources away from rAAV virion production, potentially resulting inlower rAAV virion yields.

[0146] e. Other Viral Vectors

[0147] The development and utility of viral vectors for gene delivery isconstantly improving and evolving. Other viral vectors such as poxyirus;e.g., vaccinia virus (Gnant et al., 1999; Gnant et al., 1999), alphavirus; e.g., sindbis virus, Semliki forest virus (Lundstrom, 1999),reovirus (Coffey et al., 1998) and influenza A virus (Neumann et al.,1999) are contemplated for use in the present invention and may beselected according to the requisite properties of the target system.

[0148] In certain embodiments, vaccinia viral vectors are contemplatedfor use in the present invention. Vaccinia virus is a particularlyuseful eukaryotic viral vector system for expressing heterologous genes.For example, when recombinant vaccinia virus is properly engineered, theproteins are synthesized, processed and transported to the plasmamembrane. Vaccinia viruses as gene delivery vectors have recently beendemonstrated to transfer genes to human tumor cells, e.g., EMAP-II(Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells,e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g.,P-450 (U.S. Pat. No. 5,506,138). The preparation, growth andmanipulation of vaccinia viruses are described in U.S. Pat. No.5,849,304 and U.S. Pat. No. 5,506,138 (each specifically incorporatedherein by reference).

[0149] In other embodiments, sindbis viral vectors are contemplated foruse in gene delivery. Sindbis virus is a species of the alphavirus genus(Garoff and Li, 1998) which includes such important pathogens asVenezuelan, Western and Eastern equine encephalitis viruses (Sawai etal., 1999; Mastrangelo et al., 1999). In vitro, sindbis virus infects avariety of avian, mammalian, reptilian, and amphibian cells. The genomeof sindbis virus consists of a single molecule of single-stranded RNA,11,703 nucleotides in length. The genomic RNA is infectious, is cappedat the 5′ terminus and polyadenylated at the 3′ terminus, and serves asmRNA. Translation of a vaccinia virus 26S mRNA produces a polyproteinthat is cleaved co- and post-translationally by a combination of viraland presumably host-encoded proteases to give the three virus structuralproteins, a capsid protein (C) and the two envelope glycoproteins (E1and PE2, precursors of the virion E2).

[0150] Three features of sindbis virus suggest that it would be a usefulvector for the expression of heterologous genes. First, its wide hostrange, both in nature and in the laboratory. Second, gene expressionoccurs in the cytoplasm of the host cell and is rapid and efficient.Third, temperature-sensitive mutations in RNA synthesis are availablethat may be used to modulate the expression of heterologous codingsequences by simply shifting cultures to the non-permissive temperatureat various time after infection. The growth and maintenance of sindbisvirus is known in the art (U.S. Pat. No. 5,217,879, specificallyincorporated herein by reference).

[0151] f. Chimeric Viral Vectors

[0152] Chimeric or hybrid viral vectors are being developed for use intherapeutic gene delivery and are contemplated for use in the presentinvention. Chimeric poxyiral/retroviral vectors (Holzer et al., 1999),adenoviral/retroviral vectors (Feng et al., 1997; Bilbao et al., 1997;Caplen et al., 1999) and adenoviral/adeno-associated viral vectors(Fisher et al., 1996; U.S. Pat. No. 5,871,982) have been described.

[0153] These “chimeric” viral gene transfer systems can exploit thefavorable features of two or more parent viral species. For example,Wilson et al., provide a chimeric vector construct which comprises aportion of an adenovirus, AAV 5′ and 3′ ITR sequences and a selectedtransgene, described below (U.S. Pat. No. 5,871,983, specificallyincorporate herein by reference).

[0154] The adenovirus/AAV chimeric virus uses adenovirus nucleic acidsequences as a shuttle to deliver a recombinant AAV/transgene genome toa target cell. The adenovirus nucleic acid sequences employed in thehybrid vector can range from a minimum sequence amount, which requiresthe use of a helper virus to produce the hybrid virus particle, to onlyselected deletions of adenovirus genes, which deleted gene products canbe supplied in the hybrid viral production process by a selectedpackaging cell. At a minimum, the adenovirus nucleic acid sequencesemployed in the pAdA shuttle vector are adenovirus genomic sequencesfrom which all viral genes are deleted and which contain only thoseadenovirus sequences required for packaging adenoviral genomic DNA intoa preformed capsid head. More specifically, the adenovirus sequencesemployed are the cis-acting 5′ and 3′ inverted terminal repeat (ITR)sequences of an adenovirus (which function as origins of replication)and the native 5′ packaging/enhancer domain, that contains sequencesnecessary for packaging linear Ad genomes and enhancer elements for theEl promoter. The adenovirus sequences may be modified to contain desireddeletions, substitutions, or mutations, provided that the desiredfunction is not eliminated.

[0155] The AAV sequences useful in the above chimeric vector are theviral sequences from which the rep and cap polypeptide encodingsequences are deleted. More specifically, the AAV sequences employed arethe cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. Thesechimeras are characterized by high titer transgene delivery to a hostcell and the ability to stably integrate the transgene into the hostcell chromosome (U.S. Pat. No. 5,871,983, specifically incorporateherein by reference). In the hybrid vector construct, the AAV sequencesare flanked by the selected adenovirus sequences discussed above. The 5′and 3′ AAV ITR sequences themselves flank a selected transgene sequenceand associated regulatory elements, described below. Thus, the sequenceformed by the transgene and flanking 5′ and 3′ AAV sequences may beinserted at any deletion site in the adenovirus sequences of the vector.For example, the AAV sequences are desirably inserted at the site of thedeleted E1a/E1b genes of the adenovirus. Alternatively, the AAVsequences may be inserted at an E3 deletion, E2a deletion, and so on. Ifonly the adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences areused in the hybrid virus, the AAV sequences are inserted between them.

[0156] The transgene sequence of the vector and recombinant virus can bea gene, a nucleic acid sequence or reverse transcript thereof,heterologous to the adenovirus sequence, which encodes a protein,polypeptide or peptide fragment of interest. The transgene isoperatively linked to regulatory components in a manner which permitstransgene transcription. The composition of the transgene sequence willdepend upon the use to which the resulting hybrid vector will be put.For example, one type of transgene sequence includes a therapeutic genewhich expresses a desired gene product in a host cell. These therapeuticgenes or nucleic acid sequences typically encode products foradministration and expression in a patient in vivo or ex vivo to replaceor correct an inherited or non-inherited genetic defect or treat anepigenetic disorder or disease.

[0157] E. Antibodies Against Chlamydia psittaci Proteins.

[0158] In another aspect, the present invention provides antibodycompositions that are immunoreactive with a Chlamydia psittacipolypeptide of the present invention, or any portion thereof.

[0159] An antibody can be a polyclonal or a monoclonal antibody. Anantibody may also be monovalent or bivalent. A prototype antibody is animmunoglobulin composed by four polypeptide chains, two heavy and twolight chains, held together by disulfide bonds. Each pair of heavy andlight chains forms an antigen binding site, also defined ascomplementarity-determining region (CDR). Therefore, the prototypeantibody has two CDRs, can bind two antigens, and because of thisfeature is defined bivalent. The prototype antibody can be split by avariety of biological or chemical means. Each half of the antibody canonly bind one antigen and, therefore, is defined monovalent. Means forpreparing and characterizing antibodies are well known in the art (see,e.g., Howell and Lane, 1988).

[0160] Peptides corresponding to one or more antigenic determinants of aChlamydia psittaci polypeptide of the present invention also can beprepared. Such peptides should generally be at least five or six aminoacid residues in length, will preferably be about 10, 15, 20, 25 orabout 30 amino acid residues in length, and may contain up to about35-50 residues or so. Synthetic peptides will generally be about 35residues long, which is the approximate upper length limit of automatedpeptide synthesis machines, such as those available from AppliedBiosystems (Foster City, Calif.). Longer peptides also may be prepared,e.g., by recombinant means.

[0161] The identification and preparation of epitopes from primary aminoacid sequences on the basis of hydrophilicity is taught in U.S. Pat. No.4,554,101 (Hopp), incorporated herein by reference. Through the methodsdisclosed in Hopp, one of skill in the art would be able to identifyepitopes from within an amino acid sequence such as a Chlamydia psittacipolypeptide sequence.

[0162] Numerous scientific publications have also been devoted to theprediction of secondary structure, and to the identification ofepitopes, from analyses of amino acid sequences (Chou & Fasman, 1974a;Chou & Fasman, 1974b; Chou & Fasman, 1978a; Chou & Fasman, 1978b; Chou &Fasman, 1979). Any of these may be used, if desired, to supplement theteachings of Hopp in U.S. Pat. No. 4,554,101.

[0163] Moreover, computer programs are currently available to assistwith predicting antigenic portions and epitopic core regions ofproteins. Examples include those programs based upon the Jameson-Wolfanalysis (Jameson & Wolf, 1988; Wolf et al., 1988), the program PEPPLOT®(Brutlag et al., 1990; Weinberger et al., 1985), and other new programsfor protein tertiary structure prediction (Fetrow & Bryant, 1993).Another commercially available software program capable of carrying outsuch analyses is MACVECTOR (IBI, New Haven, Conn.).

[0164] In further embodiments, major antigenic determinants of aChlamydia psittaci polypeptide may be identified by an empiricalapproach in which portions of the gene encoding the polypeptide areexpressed in a recombinant host, and the resulting proteins tested fortheir ability to elicit an immune response. For example, PCR can be usedto prepare a range of peptides lacking successively longer fragments ofthe C-terminus of the protein. The immunoactivity of each of thesepeptides is determined to identify those fragments or domains of thepolypeptide that are immunodominant. Further studies in which only asmall number of amino acids are removed at each iteration then allowsthe location of the antigenic determinants of the polypeptide to be moreprecisely determined.

[0165] Another method for determining the major antigenic determinantsof a polypeptide is the SPOTS system (Genosys Biotechnologies, Inc., TheWoodlands, Tex.). In this method, overlapping peptides are synthesizedon a cellulose membrane, which following synthesis and deprotection, isscreened using a polyclonal or monoclonal antibody. The antigenicdeterminants of the peptides which are initially identified can befurther localized by performing subsequent syntheses of smaller peptideswith larger overlaps, and by eventually replacing individual amino acidsat each position along the immunoreactive peptide.

[0166] Once one or more such analyses are completed, polypeptides areprepared that contain at least the essential features of one or moreantigenic determinants. The peptides are then employed in the generationof antisera against the polypeptide. Minigenes or gene fusions encodingthese determinants also can be constructed and inserted into expressionvectors by standard methods, for example, using PCR cloning methodology.

[0167] The use of such small peptides for antibody generation orvaccination typically requires conjugation of the peptide to animmunogenic carrier protein, such as hepatitis B surface antigen,keyhole limpet hemocyanin or bovine serum albumin. Methods forperforming this conjugation are well known in the art.

[0168] 1. Anti-Chlamydia psittaci Antibody Generation

[0169] The present invention provides monoclonal antibody compositionsthat are immunoreactive with a Chlamydia psittaci polypeptide. Asdetailed above, in addition to antibodies generated against a fulllength Chlamydia psittaci polypeptide, antibodies also may be generatedin response to smaller constructs comprising epitopic core regions,including wild-type and mutant epitopes. In other embodiments of theinvention, the use of anti-Chlamydia psittaci single chain antibodies,chimeric antibodies, diabodies and the like are contemplated.

[0170] As used herein, the term “antibody” is intended to refer broadlyto any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE.Generally, IgG and/or IgM are preferred because they are the most commonantibodies in the physiological situation and because they are mosteasily made in a laboratory setting.

[0171] Monoclonal antibodies (mAbs) are recognized to have certainadvantages, e.g., reproducibility and large-scale production, and theiruse is generally preferred.

[0172] However, “humanized” Chlamydia psittaci antibodies also arecontemplated, as are chimeric antibodies from mouse, rat, goat or otherspecies, fusion proteins, single chain antibodies, diabodies, bispecificantibodies, and other engineered antibodies and fragments thereof. Asdefined herein, a “humanized” antibody comprises constant regions from ahuman antibody gene and variable regions from a non-human antibody gene.A “chimeric antibody, comprises constant and variable regions from twogenetically distinct individuals. An anti-Chlamydia psittaci humanizedor chimeric antibody can be genetically engineered to comprise aChlamydia psittaci antigen binding site of a given of molecular weightand biological lifetime, as long as the antibody retains its Chlamydiapsittaci antigen binding site.

[0173] The term “antibody” is used to refer to any antibody-likemolecule that has an antigen binding region, and includes antibodyfragments such as Fab′, Fab, F(ab′)₂, single domain antibodies (DABs),Fv, scFv (single chain Fv), chimeras and the like. Methods andtechniques of producing the above antibody-based constructs andfragments are well known in the art (U.S. Pat. No. 5,889,157; U.S. Pat.No. 5,821,333; U.S. Pat. No. 5,888,773, each specifically incorporatedherein by reference).

[0174] U.S. Pat. No. 5,889,157 describes a humanized B3 scFv antibodypreparation. The B3 scFv is encoded from a recombinant, fused DNAmolecule, that comprises a DNA sequence encoding humanized Fv heavy andlight chain regions of a B3 antibody and a DNA sequence that encodes aneffector molecule. The effector molecule can be any agent having aparticular biological activity which is to be directed to a particulartarget cell or molecule. Described in U.S. Pat. No. 5,888,773, is thepreparation of scFv antibodies produced in eukaryotic cells, wherein thescFv antibodies are secreted from the eukaryotic cells into the cellculture medium and retain their biological activity. It is contemplatedthat similar methods for preparing multi-functional anti-Chlamydiapsittaci fusion proteins, as described above, may be utilized in thepresent invention.

[0175] Means for preparing and characterizing antibodies also are wellknown in the art (See, e.g., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference). Themethods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic Chlamydia psittaci polypeptide composition in accordancewith the present invention and collecting antisera from that immunizedanimal.

[0176] A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

[0177] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0178] As also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitablemolecule adjuvants include all acceptable immunostimulatory compounds,such as cytokines, toxins or synthetic compositions.

[0179] Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion also is contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

[0180] In addition to adjuvants, it may be desirable to coadministerbiologic response modifiers (BRM), which have been shown to upregulate Tcell immunity or downregulate suppressor cell activity. Such BRMsinclude, but are not limited to, Cimetidine (CIM; 1200 mg/d) (SmithKlineBeecham, Pa.); low-dose Cyclophosphamide (CYP; 300 mg/m²) (Johnson/Mead,N.J.), cytokines such as γ-interferon, IL-2, or IL-12 or genes encodingproteins involved in immune helper functions, such as B-7.

[0181] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization.

[0182] A second, booster injection, also may be given. The process ofboosting and titering is repeated until a suitable titer is achieved.When a desired level of immunogenicity is obtained, the immunized animalcan be bled and the serum isolated and stored, and/or the animal can beused to generate mAbs.

[0183] For production of rabbit polyclonal antibodies, the animal can bebled through an ear vein or alternatively by cardiac puncture. Theremoved blood is allowed to coagulate and then centrifuged to separateserum components from whole cells and blood clots. The serum may be usedas is for various applications or else the desired antibody fraction maybe purified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

[0184] mAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified Chlamydia psittaci polypeptide,peptide or domain, be it a wild-type or mutant composition. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells.

[0185] The methods for generating monoclonal antibodies (“Abs) generallybegin along the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells also is possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

[0186] The animals are injected with antigen, generally as describedabove. The antigen may be coupled to carrier molecules such as keyholelimpet hemocyanin if necessary. The antigen would typically be mixedwith adjuvant, such as Freund's complete or incomplete adjuvant. Boosterinjections with the same antigen would occur at approximately two-weekintervals, or the gene encoding the protein of interest can be directlyinjected.

[0187] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are selectedfor use in the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

[0188] Often, a panel of animals will have been immunized and the spleenof an animal with the highest antibody titer will be removed and thespleen lymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

[0189] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0190] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 41, Sp210-Ag14, FO, NSO/IU, MPC-11,MPCll-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

[0191] One preferred murine myeloma cell is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

[0192] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods also is appropriate (Goding pp.71-74, 1986).

[0193] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. HAT medium, a growth medium containinghypoxanthine, aminopterin and thymidine, is well known in the art as amedium for selection of hybrid cells. Aminopterin and methotrexate blockde novo synthesis of both purines and pyrimidines, whereas azaserineblocks only purine synthesis. Where aminopterin or methotrexate is used,the media is supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0194] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0195] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0196] The selected hybridomas then would be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to providemAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the mAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

[0197] mAbs produced by either means may be further purified, ifdesired, using filtration, centrifugation and various chromatographicmethods such as HPLC or affinity chromatography. Fragments of themonoclonal antibodies of the invention can be obtained from themonoclonal antibodies so produced by methods which include digestionwith enzymes, such as pepsin or papain, and/or by cleavage of disulfidebonds by chemical reduction. Alternatively, monoclonal antibodyfragments encompassed by the present invention can be synthesized usingan automated peptide synthesizer.

[0198] It also is contemplated that a molecular cloning approach may beused to generate monoclonals. For this, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 104 times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies.

[0199] Alternatively, monoclonal antibody fragments encompassed by thepresent invention can be synthesized using an automated peptidesynthesizer, or by expression of full-length gene or of gene fragmentsin, for example, E. coli.

[0200] F. Pharmaceutical Compositions

[0201] Aqueous compositions of the present invention comprise aneffective amount of a purified polynucleotide comprising a Chlamydiapsittaci sequence and/or a purified a protein, polypeptide, peptide,epitopic core region of a Chlamydia psittaci protein, and the like,dissolved and/or dispersed in a pharmaceutically acceptable carrierand/or aqueous medium. Aqueous compositions of gene therapy vectorsexpressing any of the foregoing are also contemplated.

[0202] The phrases “pharmaceutically and/or pharmacologicallyacceptable” refer to molecular entities and/or compositions that do notproduce an adverse, allergic and/or other untoward reaction whenadministered to an animal.

[0203] As used herein, “pharmaceutically acceptable carrier” includesany and/or all solvents, dispersion media, coatings, antibacterialand/or antifungal agents, isotonic and/or absorption delaying agents andthe like. The use of such media and/or agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia and/or agent is incompatible with the active ingredient, its usein the therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions. For animaland more particularly human administration, preparations should meetsterility, pyrogenicity, general safety and/or purity standards asrequired by FDA Office of Biologics standards.

[0204] The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds may generally be formulated for parenteral administration,e.g., formulated for injection via the intravenous, intramuscular,sub-cutaneous, intralesional, and/or even intraperitoneal routes, orformulated for oral or inhaled delivery. The preparation of an aqueouscompositions that contain an effective amount of purified Chlamydiapsittaci polynucleotide or polypeptide agent as an active componentand/or ingredient will be known to those of skill in the art in light ofthe present disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions and/or suspensions; solid formssuitable for using to prepare solutions and/or suspensions upon theaddition of a liquid prior to injection can also be prepared; and/or thepreparations can also be emulsified.

[0205] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions and/or dispersions; formulations includingsesame oil, peanut oil and/or aqueous propylene glycol; and/or sterilepowders for the extemporaneous preparation of sterile injectablesolutions and/or dispersions. In all cases the form must be sterileand/or must be fluid to the extent that easy syringability exists. Itmust be stable under the conditions of manufacture and/or storage and/ormust be preserved against the contaminating action of microorganisms,such as bacteria and/or fungi.

[0206] Solutions of the active compounds as free base and/orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and/ormixtures thereof and/or in oils. Under ordinary conditions of storageand/or use, these preparations contain a preservative to prevent thegrowth of microorganisms.

[0207] A Chlamydia psittaci polynucleotide or polypeptide of the presentinvention can be formulated into a composition in a neutral and/or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and/or which areformed with inorganic acids such as, for example, hydrochloric and/orphosphoric acids, and/or such organic acids as acetic, oxalic, tartaric,mandelic, and/or the like. Salts formed with the free carboxyl groupscan also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, and/or ferric hydroxides, and/or suchorganic bases as isopropylamine, trimethylamine, histidine, procaineand/or the like. In terms of using peptide therapeutics as activeingredients, the technology of U.S. Pat. Nos. 4,608,251; 4,601,903;4,599,231; 4,599,230; 4,596,792; and/or 4,578,770, each incorporatedherein by reference, may be used.

[0208] The carrier can also be a solvent and/or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and/or liquid polyethylene glycol, and/or the like),suitable mixtures thereof, and/or vegetable oils. The proper fluiditycan be maintained, for example, by the use of a coating, such aslecithin, by the maintenance of the required particle size in the caseof dispersion and/or by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and/or the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars and/or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

[0209] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and/or freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, and/or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small tumorarea.

[0210] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and/or in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and/or the like can also beemployed.

[0211] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and/orthe liquid diluent first rendered isotonic with sufficient saline and/orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and/or intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and/or either added to 1000 ml ofhypodermoclysis fluid and/or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and/or 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

[0212] A Chlamydia psittaci polynucleotide or protein-derived peptidesand/or agents may be formulated within a therapeutic mixture to compriseabout 0.0001 to 1.0 milligrams, and/or about 0.001 to 0.1 milligrams,and/or about 0.1 to 1.0 and/or even about 10 milligrams per dose and/orso. Multiple doses can also be administered.

[0213] In addition to the compounds formulated for parenteraladministration, such as intravenous and/or intramuscular injection,other pharmaceutically acceptable forms include, e.g., tablets and/orother solids for oral administration; liposomal formulations; timerelease capsules; and/or any other form currently used, includingcremes.

[0214] One may also use nasal solutions and/or sprays, aerosols and/orinhalants in the present invention. Nasal solutions are usually aqueoussolutions designed to be administered to the nasal passages in dropsand/or sprays. Nasal solutions are prepared so that they are similar inmany respects to nasal secretions, so that normal ciliary action ismaintained. Thus, the aqueous nasal solutions usually are isotonicand/or slightly buffered to maintain a pH of 5.5 to 6.5. In addition,antimicrobial preservatives, similar to those used in ophthalmicpreparations, and/or appropriate drug stabilizers, if required, may beincluded in the formulation. Various commercial nasal preparations areknown and/or include, for example, antibiotics and/or antihistaminesand/or are used for asthma prophylaxis.

[0215] Additional formulations which are suitable for other modes ofadministration include vaginal suppositories and/or pessaries. A rectalpessary and/or suppository may also be used. Suppositories are soliddosage forms of various weights and/or shapes, usually medicated, forinsertion into the rectum, vagina and/or the urethra. After insertion,suppositories soften, melt and/or dissolve in the cavity fluids. Ingeneral, for suppositories, traditional binders and/or carriers mayinclude, for example, polyalkylene glycols and/or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%.

[0216] Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand/or the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulationsand/or powders. In certain defined embodiments, oral pharmaceuticalcompositions will comprise an inert diluent and/or assimilable ediblecarrier, and/or they may be enclosed in hard and/or soft shell gelatincapsule, and/or they may be compressed into tablets, and/or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand/or used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and/or the like. Suchcompositions and/or preparations should contain at least 0.1% of activecompound. The percentage of the compositions and/or preparations may, ofcourse, be varied and/or may conveniently be between about 2 to about75% of the weight of the unit, and/or preferably between 25-60%. Theamount of active compounds in such therapeutically useful compositionsis such that a suitable dosage will be obtained.

[0217] The tablets, troches, pills, capsules and/or the like may alsocontain the following: a binder, as gum tragacanth, acacia, cornstarch,and/or gelatin; excipients, such as dicalcium phosphate; adisintegrating agent, such as corn starch, potato starch, alginic acidand/or the like; a lubricant, such as magnesium stearate; and/or asweetening agent, such as sucrose, lactose and/or saccharin may be addedand/or a flavoring agent, such as peppermint, oil of wintergreen, and/orcherry flavoring. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carrier.Various other materials may be present as coatings and/or to otherwisemodify the physical form of the dosage unit. For instance, tablets,pills, and/or capsules may be coated with shellac, sugar and/or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and/or propylparabens as preservatives, a dye and/orflavoring, such as cherry and/or orange flavor.

[0218] G. Kits

[0219] Therapeutic kits of the present invention are kits comprising aChlamydia psittaci polynucleotide or polypeptide. Such kits willgenerally contain, in suitable container means, a pharmaceuticallyacceptable formulation of a Chlamydia psittaci polynucleotide orpolypeptide or vector expressing any of the foregoing in apharmaceutically acceptable formulation. The kit may have a singlecontainer means, and/or it may have distinct container means for eachcompound.

[0220] When the components of the kit are provided in one and/or moreliquid solutions, the liquid solution is an aqueous solution, with asterile aqueous solution being particularly preferred. The Chlamydiapsittaci polynucleotide or polypeptide compositions may also beformulated into a syringeable composition. In which case, the containermeans may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit.

[0221] However, the components of the kit may be provided as driedpowder(s). When reagents and/or components are provided as a dry powder,the powder can be reconstituted by the addition of a suitable solvent.It is envisioned that the solvent may also be provided in anothercontainer means.

[0222] The container means will generally include at least one vial,test tube, flask, bottle, syringe and/or other container means, intowhich the Chlamydia psittaci polynucleotide or polypeptide formulationare placed, preferably, suitably allocated. The kits may also comprise asecond container means for containing a sterile, pharmaceuticallyacceptable buffer and/or other diluent.

[0223] The kits of the present invention will also typically include ameans for containing the vials in close confinement for commercial sale,such as, e.g., injection and/or blow-molded plastic containers intowhich the desired vials are retained.

[0224] Irrespective of the number and/or type of containers, the kits ofthe invention may also comprise, and/or be packaged with, an instrumentfor assisting with the injection/administration and/or placement of theultimate Chlamydia psittaci polynucleotide or polypeptide within thebody of an animal. Such an instrument may be a syringe, pipette,forceps, and/or any such medically approved delivery vehicle.

H. EXAMPLES

[0225] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Exemplary ELI Protocol

[0226] The following sections outline general methodology that one mightuse to prepare, screen and utilize ELI according to the presentinvention. Of course the following methods are merely general guidelinesand should not limit one of skill in the art from modifying the presentinvention to accomplish a desired goal using ELI.

[0227] 1. Library Construction

[0228] The present invention provides expression library constructs ofgenus Chlamydia psittaci. An expression library of C. psittaci can beproduced by first physically shearing the genomic DNA of C. psittaci(e.g., C. psittaci strain B577) and size-selecting fragments of 300-800base pairs. The protocol used by the present inventors to produce a C.psittaci library is similar to that described in Sykes and Johnston(1999). Adaptors were added and the DNA fragments ligated into a geneticimmunization vector (FIG. 2) designed to link fragments to the mouseubiquitin gene. However, the fragments can be blunt-end cloned.

[0229] This vector is known to enhance MHC class I-restricted immuneresponses (Sykes and Johnston, 1999), while sterilizing immunity againstChlamydia is thought to be MHC class II-dependent (Morrison et al.,1995). However, any genetic immunization procedure, by the mechanism ofintracellular expression of the inserted genes, will target towardsclass I antigen presentation. Nevertheless, both MHC class I- and classII-restricted immune responses to the expressed antigens are welldocumented (Barry et al., 1995; Sykes and Johnston, 1999). The inventorsobserved, for instance, pronounced delayed-type hypersensitivityresponses, mediated by MHC II-restricted CD4⁺ Th1 cells, againstprotective C. psittaci B577 antigens, which were expressed from theubiquitin fusion vector. In addition to the fact that MHC II-restrictedimmunity is generated by the ubiquitin fusion vector, MHC I-restrictedimmunity appears to mediate protection in the early phase of chlamydialinfection (Morrison et al., 1995; Rottenberg et al., 1999). This dualityof the cellular immune response generated by the ubiquitin fusion vectormight explain the efficacy of this vector for genetic immunizationagainst intracellular bacteria.

[0230] A library of approximately 82,000 individual members was createdand tested as 27 sub-libraries each with 2,400-3,400 plasmid clones. Theaverage insert frequency was approximately 67% and the average insertsize was 660 base pairs. Nitrocellulose replica filters were made ofeach original colony plating of a sub-library pool for subsequentretrieval of positive clones. This generated a library withapproximately six-fold expression-equivalent redundancy. One expressionequivalent is defined as the number of in-frame fragments necessary tocompletely represent all authentic open reading frames. Since the genomesize of C. psittaci is approximately 1×10⁶base pairs and only one-sixthof the actual open-reading frames will be cloned in the rightorientation and frame, it requires at least six genomic equivalents toencode one expression equivalent. Each sub-library was propagated onplates and harvested to prepare DNA. DNA representing each sub-librarywas used for genetic immunization of mice in the following section.

[0231] 2. Vaccination and Challenge

[0232] For the first round of testing, outbred, 6-week old, femaleN1H-Swiss Webster mice were inoculated with the purified DNA of eachsub-library using both intra-muscular (i.m.) and epidermal injection.The epidermal injection was effected with a gene gun (Sanford et al.,1991). Each mouse was given 50 μg DNA i.m. and 5 μg DNA by gene gun. Ithas been argued that the gene gun immunization favors a Th2 and the i.m.injection a Th1 type response (Feltquate et al., 1997), therefore bothtypes of injection were given to each group. In the first round oftesting, the prime inoculation was followed by a boost 9 weeks later,before intranasal challenge with 3×10⁶ inclusion forming units (IFU) ofC. psittaci strain B577 13 weeks after prime inoculation. The animalswere sacrificed 12 days after the challenge, and lungs were weighed.

[0233] 3. Library Deconvolution

[0234] The basic scheme for handling the reduction of the libraries isdepicted in FIG. 3. Fourteen groups out of the first round lookedpromising, so the individual clones from these groups were picked andgrown in 96 well microtiter plates. This gave approximately 40,000 wellsin microtiter plates, therefore about 40,000 clones. The second roundwas reduced using a two dimensional array format. As depicted in FIG. 3,the DNA was prepared from colonies pooled from rows and columns of thearray. The rationale was that if a row and column conferred protection,the colonies at the intersection would be responsible. This scheme ispremised on largely additive effects of the protective clones. This24×24 array yielded pools of 1,700 clones with each intercession having˜96 clones. Currently the inventors deconvolute the second round with a3-dimesional array.

[0235] Since the lung weight was highly variable in the outbredN1H-Swiss mice with variable MHC background, the inventors decided touse inbred BALB/c mice in subsequent rounds. The 48 DNA pools for roundtwo were i.m. injected into BALB/c mice at 50 μg DNA/animal, and theanimals were boosted at seven weeks by both gene gun inoculation andi.m. injection. The mice were given a higher C. psittaci challenge,1.6×10⁶ IFU C. psittaci B577, at approximately 12 weeks, again tofurther differentiate the groups. Animals were sacrificed and resultsevaluated as in round one.

[0236] In the fourth round, the animals received two boosts rather thanone, and the challenge inoculum was increased to 3×10⁶ IFU C. psittaciB577 to increase the selectivity of protection scoring. Furthermore,because too much DNA may lead to a decrease in cellular immune response,the amount of each individual clone was reduced by half, with thedifference made up with pUC 118 DNA, so each mouse received a total 50μg DNA for i.m. immunization, but only 25 μg of the specific clone. Theinventors also decreased the gene gun DNA in the same manner: 1.25μg/ear of the specific clone and 1.25 μg pUC118. Mice were boosted i.m.at both four and nine weeks after prime inoculation, and werechallenged. The results of this final round are depicted in FIG. 5.

[0237] 4. Analysis of Sequences

[0238] The clones conferring protection were re-sequenced and thencompared by BLAST search to Genbank and particularly to the recentlycompleted C. pneumoniae (Kalman et al., 1999) genome sequences (FIG. 6).Of the 14 single genes identified in this study, ten are internalfragments and three contain the C-terminus of the protein. Of the fivemost protective clones, one was from a putative outer membrane proteinand one was from a cell surface protein. The other three were fromcytosolic proteins.

[0239] Four of the 14 clones have sequence similarity to a class ofproteins known as putative outer membrane proteins (POMPs) in Chlamydiapsittaci and Chlamydia pneumoniae. Many of the “putative” outer membraneproteins are known to be localized to the outer membrane and to behighly immunogenic (Longbottom et al., 1996; Tan et al., 1990).

[0240] 5. Mixing Experiment

[0241] The two dimensional approach used to find protective genefragments assumes that the protection is due to a single highlyprotective gene within a pool. To verify that such genes would be found,25 ng (i.e. {fraction (1/2000)}) of either of the two most protectivegenes was added to a pool that scored negative (pool 6 round 1). Asdepicted in FIG. 7, spiking with either clone converted the negativelibrary to a positive.

Example 2 Materials and Methods

[0242] Library construction. C. psittaci strain B577 (ATCC VR-656) wasgrown in BGMK cells and elementary bodies (EB) were purified byrenograff gradient centrifugation as described (Huang et al., 1999).Genomic DNA was isolated from EB by proteinase K and RNase digestionfollowed by cetyl-trimethyl ammonium bromide (Kaltenbock et al., 1997).

[0243] Genomic DNA was physically sheared using a nebulizer (Glas Col,Terra Haute, Ind.), then size fractioned on a 1.5% TBE agarose gel.Agarose with fragments between 300-700 base pairs was excised and theDNA was electroeluted. Adaptors (top strand 5′: GATCTGGATCCCGAT (SEQ IDNO.2) ATCGGGCTCCA (SEQ ID NO.3) onto the fragments, then the fragmentswere cloned into pCMVi-UBs at the Bgl II site (See FIG. 6 and Sykes andJohnston, 1999 for more details). The ligations were transformed into DHalpha electrocompetent cells and plated onto 150 mm diameterYT-Ampicillin (75 μg/mL final concentration) plates. The resultingplates had between 2400-3400 individual clones per plate. After theplates were incubated overnight at 37° C., the colonies from were liftedusing nitrocellulose filters soaked in L-Broth with 8% DMSO, and thesefilters were stored at −80° C. The original agar plates were thenincubated at 37° C. for an additional six hours. Ten mL of L Broth wasadded to each plate, the E. coli was scraped into 150 mL of L-Broth andgrown at 37° C. for 30 minutes. Ampicillin was then added to a finalconcentration of 50 μg/mL, and the cultures were grown overnight at 37°C. Cells were pelleted and the DNA was purified using Qiagen tip 500columns.

[0244] Inoculation of DNA. Round One: DNA from the pools was injectedinto 6-week old female N1H-Swiss mice. All mice received 50 μg total DNAby i.m. injections, evenly distributed between the quadriceps andtibialis anterior muscles. Eighteen of the groups also received gene guninoculations (wand), with 2.5 μg DNA inoculated into each ear. Theanimals were boosted once at nine weeks in the same manner as theprimary inoculation—all mice received i.m. injections, but only the same18 groups received gene gun injections—then intranasally challenged with5.5×10⁵ IFU of C. psittaci strain B577 at 13 weeks. The mice weresacrificed 11 days after the challenge, and lungs were weighed.

[0245] Round Two: Nitrocellulose filters from the positive pools wereplaced on L-Broth Bio-Assay plates supplemented with 75 μg/mL ampicillinand 2% agar. The filters were incubated on the plates for approximately15 minutes, then the nitrocellulose was discarded. The colonies weregrown at 30° C. for 12 hours. The majority of the colonies were pickedinto 96 well microtiter plates containing HYT media (1.6%Bacto-tryptone, 1.0% Bacto-yeast extract, 85.5 mM NaCl, 36 mM K₂HPO₄,13.2 mM KH₂PO₄, 1.7 mM Sodium citrate, 0.4 mM MgSO4, 6.8 mM ammoniumsulfate, 4.4% wt/vol glycerol) supplemented with 75 μg/mL ampicillin,using a Hybaid colony picker; the plates were then visually inspectedand the remainder of the colonies were hand-picked. The microtiterplates were designated by their original pool number and by the order inwhich they were picked. Hence, plate 5.10 was from original pool 5 andwas the tenth plate picked. The colonies were subdivided into groups asis indicated in FIG. 2. All of the microtiter plates comprising a poolwere stamped onto on L-Broth Bio-Assay plates supplemented with 75 μg/mLampicillin and were grown overnight at 37° C. The cells from theseplates were harvested by adding L-Broth to the plates and scraping offthe cells. The cells were pelleted by centrifugation then resuspended inQiagen buffer P1. The remainder of the DNA prep proceeded according tomanufacture's instructions.

[0246] These 48 DNA pools were i.m. injected into 6-week old BALB/c miceat 50 μg DNA/animal. For the initial inoculation, the mice did notreceive gene gun inoculations. At seven weeks, the mice were boostedwith 50 μg DNA/animal. In addition to the i.m. injections, the first 31groups received gene gun (Rumsey-Loomis) inoculations at 2.5 μg DNA/ear;however, the gene gun failed at group 32, and the last 17 groupsreceived only i.m. injections. The mice were given a higher challenge,1.6×10⁶ IFU C. psittaci B577, at 12 weeks. Animals were sacrificed as inround one.

[0247] Round Three: Colonies from the microtiter plates that were judgedto be positive were arrayed as in FIG. 2. For each pool, new microtiterplates with HYT media supplemented with 75 μg/mL ampicillin wereconstructed from all of the colonies which comprise the. Colonies weregrown and DNA prepared as in round two.

[0248] The mice received both gene gun (wand) and i.m. inoculations atthe dosage indicated above. At six weeks, the mice were boosted with 50μg DNA/animal, but only by i.m. injections. The challenge schedule wasthe same as in Round Two.

[0249] Round Four: E. coli from wells at either full by full protectionor full by partial protection was streaked out onto YT-platessupplemented with 75 μg/mL ampicillin. Six colonies from each of theplates were tested by PCR colony screening, using the primers FS-UB 5′:CCGCACCCTCTCTGATTAC (SEQ ID No: 4) CTGGAGTGGCAACTTCC. (SEQ ID NO. 5)Colonies with different sizes, hence different inserts, were sequencedusing ABI Big Dye terminator and the FS-UB primer. Samples were purifiedon G-50 spin columns, and run on an ABI 377 Sequencer. The generatedsequences were analyzed for open reading frames using a program designedby Simon Raynor, Ph.D.

Example 3 Vaccination and Challenge

[0250] It was established that the weight increase of the infected lungover the lung weight of naive, uninfected controls (˜120 mg) correlatedstrongly with disease intensity. Maximum disease in this model resultedin approximately 250% lung weight increase, while further lung weightincreases were lethal. The lung disease on day 12 after inoculation wascharacterized by areas of gross lung tissue consolidation and thepresence of mononuclear interstitial infiltrates in consolidated tissue.Chlamydial inclusions were observed by immunohistochemistry in manymacrophages, but rarely in other cells. Controls for complete protectionwere established by low level intranasal infection of naïve mice with3×10⁴ IFU of C. psittaci strain B5774 weeks prior to challenge. Thesemice were completely protected from disease after challenge infectionand had lung weight increases of 10-30% compared to naive animals. Lungsof completely protected mice did not show gross lung lesions, andpathohistological examination revealed no interstitial infiltrates, butprominent peribronchiolar lymphocytic cuffs, interpreted as sign ofprotective immune stimulation. The chlamydial lung burden on day 11after challenge was typically 1-3×10⁶ IFU per 100 mg lung tissue inprotected, and 2-6×10⁶ IFU per 100 mg lung in diseased animals. Sincethe lowest chlamydial burden was, however, not consistently associatedwith lowest disease, the inventors used the disease-dependent parameterlung weight rather than chlamydial burden as readout for evaluation ofprotection. The lung weights were transformed to relative protectionscores in a linear equation that assumed the high average lung weight ofthe severely ill, naïve, challenged mice as 0 and that of fullyprotected controls as 1 (FIG. 4).

Example 4 Deconvolution of the Libraries

[0251] Since the lung weight was highly variable in the outbredN1H-Swiss mice with variable MHC background, the inventors decided touse inbred BALB/c mice in subsequent rounds. The 48 DNA pools for roundtwo were i.m. injected into BALB/c mice at 50 μg DNA/animal, and theanimals were boosted at seven weeks by both gene gun inoculation andi.m. injection. The mice were given a higher C. psittaci challenge,1.6×10⁶ IFU C. psittaci B577, at approximately 12 weeks, again tofurther differentiate the groups. Animals were sacrificed and resultsevaluated as in round one.

[0252] The results of the Round two challenge are depicted in FIG. 4. Ofthe 48 groups from round two, 15 were judged to be positive, giving atotal of 3936 wells. These wells were again arrayed as in round two, butthe array had 112 colonies per column and 156 per row with 4-5 coloniesper intersection (See FIG. 3). The mice received both gene gun and i.m.injections at the dosage indicated above. At six weeks, the mice wereboosted. Both the challenge and the sacrifice were performed as in Roundtwo.

[0253] The positive 46 colonies from the intersection wells from Roundthree were sequenced, and those clones with open reading frames greaterthan 50 amino acids long were prepared individually and shot into miceas single genes and as a pool. Fourteen clones met these criteria. Thedisease scoring on each pool in rounds 1-3 are depicted in FIG. 4.

[0254] In the fourth round, the animals received two boosts rather thanone, and the challenge inoculum was increased to 3×10⁶ IFU C. psittaciB577 to increase the selectivity of protection scoring. Furthermore,because too much DNA may lead to a decrease in cellular immune response,the amount of each individual clone was reduced by half but made up thedifference with pUC 118 DNA, and each mouse received a total 50 μg DNAfor i.m. immunization, but only 25 μg of the specific clone. Theinventors also decreased the gene gun DNA in the same manner: 1.25μg/ear of the specific clone and 1.25 μg pUCI18. Mice were boosted i.m.at both four and nine weeks after prime inoculation, and werechallenged. The results of this final round are depicted in FIG. 5.

Example 5 Comparison of Clones

[0255] Based on the hypothesis that sequences from genes conferring ahigh level of protection might be selected more than once in the ELIprocess, the clones were compared against each other for overlaps.Interestingly, one of the clones, CP4 #10, did overlap with anothergene, CP4 #11. The gene from which these two clones arise had beenpartially sequenced (Longbottom et al., 1998).

[0256] Two of the genes, CP4 #5 and CP4 #9, had an overlapping region,but they were fused to ubiqutin in opposite orientations. CP4 #5, iscomposed of two different C. psittaci DNA fragments, fused in oppositeorientations. The first gene is fused to ubiqutin in the correctorientation and the correct reading frame. Interestingly, the secondgene, which is in the opposite orientation to the ubiqutin gene, has anoverlapping sequence to CP4 #5. It is doubtful that the protein from thesecond gene is produced in the mouse.

Example 6 Analysis of Sequences

[0257] The clones conferring protection were re-sequenced and thencompared by BLAST search to Genbank and particularly to the recentlycompleted C. pneumoniae (Kalman et al., 1999) genome sequences (FIG. 6).The full-length Chlamydia psittaci genes were next isolated andsequences. Upon analysis, all nucleic acid sequences, except #4, #10,#11, and #12, were previously undisclosed in any context. Further, onlyprotions of the sequences encoding #10 and #11 were previouslydisclosed.

[0258] Since most protective genes would not have been predicted by anybioinformatics or information-based approach, it is likely that one willneed to apply an unbiased, global approach, such as ELI to definevaccine candidates.

[0259] Table 2, lists a comparision of the Chlamydia psittaci genes withhomologues from Chlamydia trachomatis and Chlamydia pneumoniae. TABLE 2C. psittaci C. trachomatis Identity/Similarity C. pneumoniaeIdentity/Similarity CP4 #1 DNA Pol III Gamma and Tau 62/73 DNA Pol IIIGamma and Tau 66/76 CP4 #2 Glu-tRNA Gln Amido- 49/70 Glu-tRNA Gln Amido-48/63 transferase (C subunit) transferase (C subunit) CP4 #3 Glu-tRNAGln Amido- 71/85 Glu-tRNA Gln Amido- 71/84 transferase (A subunit)transferase (A subunit) CP4 #4 OMP 90A Outer Membrane Protein 5 30/45Outer Membrane Protein I 40/54 CP4 #5 Transglycolase/transpeptidase67/80 Transglycolase/transpeptidase 67/77 CP4 #6 Protein Translocase80/89 Protein Translocase 84/92 CP4 #7 Outer Membrane Lipoprotein 60/79CP4 #8 Oligopeptidase 60/75 Oligopeptidase 61/74 CP4 #9 Hypotheticalprotein 62/76 Hypothetical protein 62/77 CP4 #10 Outer Membrane Protein4 27/42 Outer Membrane Protein G 33/51 CP4 #11 Outer Membrane Protein 427/42 Outer Membrane Protein G 33/51 CP4 #12 OMP 98 kDa Outer MembraneProtein 5 30/43 Outer membrane Protein G 44/58 CP4 #13 ProteinTranslocase 80/89 Protein Translocase 84/92 CP4 #14 SuccinateDehydrogenase 60/76 Succinate Dehydrogenase 61/77

[0260] Table 3 lists all of the cloned fragments, their corresponsingfull length nucleotide sequences, and the amino acid sequences encodedby both the fragments and the full length sequences. Table 2 furtherdescribes the fragments. TABLE 3 SEQUENCE LISTING INDEX SEQ ID NO CP4 NODescription SEQ ID NO: 6 CP4 #1 (fragment) homolog to C. pneumoniae DNAPol III Gamma and Tau subunits (dnaX2 gene) SEQ ID NO: 7 CP4 #1Polypeptide translation corresponding to SEQ ID NO. 6, homolog to C.pneumoniae DNA Pol III Gamma and Tau subunits (dnaX2 gene) SEQ ID NO: 8CP4 #1 (full length) homolog to C. pneumoniae DNA Pol III Gamma and Tausubunits (dnaX2 gene) SEQ ID NO: 9 CP4 #1 Polypeptide translationcorresponding to SEQ ID NO. 8, homolog to C. pneumoniae DNA Pol IIIGamma and Tau subunits (dnaX2 gene) SEQ ID NO: 10 CP4 #2 (fragment)homolog to C. pneumoniae Glu-tRNA Gln Amido-transferase (C subunit)(gatC gene) SEQ ID NO: 11 CP4 #2 Polypeptide translation correspondingto SEQ ID NO. 10, homolog to C. pneumoniae Glu-tRNA GlnAmido-transferase (C subunit) (gatC gene) SEQ ID NO: 12 CP4 #2 (fulllength) homolog to C. pneumoniae Glu-tRNA Gln Amido-transferase (Csubunit) (gatC gene) SEQ ID NO: 13 CP4 #2 Polypeptide translationcorresponding to SEQ ID NO. 12, homolog to C. pneumoniae Glu-tRNA GlnAmido-transferase (C subunit) (gatC gene) SEQ ID NO: 14 CP4 #3(fragment) homolog to C. pneumoniae Glu-tRNA Gln Amido-transferase (Asubunit) (gatA gene) SEQ ID NO: 15 CP4 #3 Polypeptide translationcorresponding to SEQ ID NO. 14, homolog to C. pneumoniae Glu-tRNA GlnAmido-transferase (A subunit) (gatA gene) SEQ ID NO: 16 CP4 #3 (fulllength) homolog to C. pneumoniae Glu-tRNA Gln Amido-transferase (Asubunit) (gatA gene) SEQ ID NO: 17 CP4 #3 Polypeptide translationcorresponding to SEQ ID NO. 16, homolog to C. pneumoniae Glu-tRNA GlnAmido-transferase (A subunit) (gatA gene) SEQ ID NO: 18 CP4 #3 (fulllength) homolog to C. pneumoniae Glu-tRNA Gln Amido-transferase (Bsubunit) (gatB gene) SEQ ID NO: 19 CP4 #3 Polypeptide translationcorresponding to SEQ ID NO. 18, homolog to C. pneumoniae Glu-Trna GlnAmido-transferase (B subunit) (gatB gene) SEQ ID NO: 20 CP4 #4(fragment) C. psittaci 90 kDa outer membrane protein (OMP90A gene)(Previously sequenced by Longbottom, et al) SEQ ID NO: 21 CP4 #4Polypeptide translation corresponding to SEQ ID NO. 20, C. psittaci 90kDa outer membrane protein (OMP90A gene) SEQ ID NO: 22 CP4 #4 (fulllength) C. psittaci 90 kDa outer membrane protein (OMP90A gene)(Previously sequenced by Longbottom, et al) SEQ ID NO: 23 CP4 #4Polypeptide translation corresponding to SEQ ID NO. 22, C. psittaci 90kDa outer membrane protein (OMP90A gene) SEQ ID NO: 24 CP4 #5 (fragment)homolog to C. pneumoniae transglycolase/transpeptidase (pbp3 gene) SEQID NO: 25 CP4 #5 Polypeptide translation corresponding to SEQ ID NO. 24,homolog to C. pneumoniae transglycolase/transpeptidase (pbp3 gene) SEQID NO: 26 CP4 #5 (full length) homolog to C. pneumoniaetransglycolase/transpeptidase (pbp3 gene) SEQ ID NO: 27 CP4 #5Polypeptide translation corresponding to SEQ ID NO. 26, homolog to C.pneumoniae transglycolase/transpeptidase (pbp3 gene) SEQ ID NO: 28 CP4#6 (fragment) homolog to C. pneumoniae Protein Translocase (secA2 gene)SEQ ID NO: 29 CP4 #6 Polypeptide translation corresponding to SEQ ID NO.28, homolog to C. pneumoniae Protein Translocase (secA2 gene) SEQ ID NO:30 CP4 #13 (fragment) homolog to C. pneumoniae Protein Translocase(secA2 gene) SEQ ID NO: 31 CP4 #13 Polypeptide translation correspondingto SEQ ID NO. 30, homolog to C. pneumoniae Protein Translocase (secA2gene) SEQ ID NO: 32 CP4 #6 & 13 (full length) homolog to C. pneumoniaeProtein Translocase (secA2 gene) SEQ ID NO: 33 CP4 #6 & 13 Polypeptidetranslation corresponding to SEQ ID NO. 32, homolog to C. pneumoniaeProtein Translocase (secA2 gene) SEQ ID NO: 34 CP4 #7 (fragment) homologto C. pneumoniae Outer Membrane Lipoprotein (Cpn 0278) SEQ ID NO: 35 CP4#7 Polypeptide translation corresponding to SEQ ID NO. 34, homolog to C.pneumoniae Outer Membrane Lipoprotein (Cpn 0278 gene) SEQ ID NO: 36 CP4#7 (full length) homolog to C. pneumoniae Outer Membrane Lipoprotein(Cpn 0278) SEQ ID NO: 37 CP4 #7 Polypeptide translation corresponding toSEQ ID NO. 36, homolog to C. pneumoniae Outer Membrane Lipoprotein (Cpn0278 gene) SEQ ID NO: 38 CP4 #8 (fragment) homolog to C. pneumoniaeOligopeptidase (pepF gene) SEQ ID NO: 39 CP4 #8 Polypeptide translationcorresponding to SEQ ID NO. 38, homolog to C. pneumoniae Oligopeptidase(pepF gene) SEQ ID NO: 40 CP4 #8 (fragment) homolog to C. pneumoniaeOligopeptidase (pepF gene) SEQ ID NO: 41 CP4 #8 Polypeptide translationcorresponding to SEQ ID NO. 40, homolog to C. pneumoniae Oligopeptidase(pepF gene) SEQ ID NO: 42 CP4 #9 (fragment) homolog to C. pneumoniaegene of unknown function, co-translationaly coupled to Yop NFlagellar-Type ATPase (Cpn 0708 gene) SEQ ID NO: 43 CP4 #9 Polypeptidetranslation corresponding to SEQ ID NO. 42, homolog to C. pneumoniaegene of unknown function, co-translationally coupled to Yop NFlagellar-Type ATPase (Cpn 0708 gene) SEQ ID NO: 44 CP4 #9 (full length)homolog to C. pneumoniae gene of unknown function, co-translationallycoupled to Yop N Flagellar-Type ATPase (Cpn 0708 gene) SEQ ID NO: 45 CP4#9 Polypeptide translation corresponding to SEQ ID NO. 44, homolog to C.pneumoniae gene of unknown function, co-translationally coupled to Yop NFlagellar-Type ATPase (Cpn 0708 gene) SEQ ID NO: 46 CP4 #9 (full length)homolog to C. pneumoniae Yop N Flagellar-Type ATPase (yscN gene) SEQ IDNO: 47 CP4 #9 Polypeptide translation corresponding to SEQ ID NO. 46,homolog to C. pneumoniae Yop N Flagellar-Type ATPase (yscN gene) SEQ IDNO: 48 CP4 #10 (fragment) homolog to C. pneumoniae outer membraneprotein G (pmp 2 gene) (Nucleotides 1-423 were previously sequenced byLongbottom et al.) SEQ ID NO: 49 CP4 #10 Polypeptide translationcorresponding to SEQ ID NO. 48, homolog to C. pneumoniae outer membraneprotein G (pmp 2 gene) SEQ ID NO: 50 CP4 #11 (fragment) homolog to C.pneumoniae outer membrane protein G (pmp 2 gene) (Nucleotides 1-301 werepreviously sequenced by Longbottom et al.) SEQ ID NO: 51 CP4 #11Polypeptide translation corresponding to SEQ ID NO. 50, homolog to C.pneumoniae outer membrane protein G (pmp 2 gene) SEQ ID NO: 52 CP4 #10 &(full length) homolog to C. pneumoniae outer 11 membrane protein G (pmp2 gene). This gene immediately follows the OMP90A gene on C. psittaci,and nucleotides 1-502 were published by Longbottom et al., although theydid not report this as a gene. SEQ ID NO: 53 CP4 #10 & Polypeptidetranslation corresponding to SEQ ID 11 NO. 52, homolog to C. pneumoniaeouter membrane protein G (pmp 2 gene). SEQ ID NO: 54 CP4 #12 (fragment)C. psittaci 98 kDa outer membrane protein (POMP gene) (Previouslysequenced by Longbottom, et al) SEQ ID NO: 55 CP4 #12 Polypeptidetranslation corresponding to SEQ ID NO. 54, C. psittaci 98 kDa outermembrane protein (POMP gene) SEQ ID NO: 56 CP4 #12 (full length) C.psittaci 98 kDa outer membrane protein (POMP gene) (Previously sequencedby Longbottom et al.) SEQ ID NO: 57 CP4 #12 Polypeptide translationcorresponding to SEQ ID NO. 56, C. psittaci 98 kDa outer membraneprotein (POMP gene) SEQ ID NO: 58 CP4 #14 (fragment) homolog to C.pneumoniae Succinate Dehydrogenase (sdhC) SEQ ID NO: 59 CP4 #14Polypeptide translation corresponding to SEQ ID NO. 58, homolog to C.pneumoniae Succinate Dehydrogenase (sdhC gene) SEQ ID NO: 60 CP4 #14(full length) homolog to C. pneumoniae Succinate Dehydrogenase (sdhC)SEQ ID NO: 61 CP4 #14 Polypeptide translation corresponding to SEQ IDNO. 60, homolog to C. pneumoniae Succinate Dehydrogenase (sdhC gene)

[0261] Of the 14 single genes identified in this study, ten are internalfragments and three contain the C-terminus of the protein. Of the fivemost protective clones (CP4 #1-5), one was from a putative outermembrane protein (CP4 #4) and one was from a cell surface protein (CP4#5). The other three were from cytosolic proteins, with CP4 #2 and CP4#3 deriving independently from genes encoding a particularamidotransferase complex.

[0262] Four of the 14 clones have sequence similarity to a class ofproteins known as putative outer membrane proteins (POMPs) in Chlamydiapsittaci and Chlamydia pneumoniae (CP4 #4, CP4 #10, CP4 #11 and CP4#12). Many of the “putative” outer membrane proteins are known to belocalized to the outer membrane and to be highly immunogenic (Longbottomet al., 1996; Tan et al., 1990). The clone designated CP4 #4 is anin-frame fragment of POMP90A (Longbottom et al., 1998) and CP4 #12 is anin-frame fragment of a 98 kDa POMP which has been completely sequenced(Accession U72499). The clones CP4 #10 and CP4 #11 immediately followCP4 #4 in the genome and have sequence similarity to POMPs in C.psittaci, C. trachomatis and C. pneumoniae. As stated earlier, the cloneCP4 #10 overlaps the CP4 #11 clone. Of these clones only CP4 #4 conferssignificant protection in isolation so clearly the criteria of being anouter membrane protein is not sufficient to predict a protectivevaccine.

Example 7 Mixing Experiment

[0263] The two dimensional approach used to find protective genefragments assumes that the protection is due to a single highlyprotective gene within a pool. To verify that such genes would be found,25 ng (i.e. {fraction (1/2000)}) of either CP4 #4 or CP4 #11 was addedto a pool that scored negative (pool 6 round 1). As depicted in FIG. 7,spiking with either clone converted the negative library to a positive.Of note is that CP4 #11 did not confer protection when testedindividually, however, it does protect in combination.

[0264] The fact that a CP4 #4 positive library confers protectionvalidates the sensitivity of the system. The fact that a CP4 #11positive library protects implies that CP4 #11 con be a useful componentof a vaccine, but that it may depend upon having other antigens present.A likely explaination is that CP4 #11 is a good vaccine antigen, butrequires immunological help.

Example 8 Vaccination in Cattle

[0265] An important question is whether the genes identified in thismanner in a mouse model are clinically relevant. Of course, this concernis not peculiar to genetic vaccines or ELI, but any system that usesmodels to identify vaccine candidates. In this case the clinicallyrelevant situation is protection of cattle. In a preliminary experiment,the inventors evaluated the pool of 14 individual clones in the originalhost in a fertility challenge model. All fourteen clones were used asthe individual test data on each clone in mice was not available by thetime it was necessary to initiate the cow trial. TABLE 3. TABLE 3 C.psittaci Vaccine in Cows Percent Not Pregnant Pregnant Pregnant NotChallenged 75 3 1 Challenged, 0 0 4 Not Vaccinated EB Vaccine 25 1 3Genetic 33 2 4 Vaccine (14 gene pool)

[0266]C. psittaci is normally introduced by the fecal-oral andrespiratory routes in cattle, and disseminates to other tissuesincluding reproductive organs. C. psittaci infection of the uterinemucosa reduces fertility, the basis of the economic interest in a C.psittaci vaccine. Four groups of heifers were used. One group was thenaive unchallenged control, another the naive, challenged control, athird received the same pool of fourteen gene fragments that were testedin mice, and the fourth group was vaccinated with an experimental,inactivated vaccine of elementary bodies (EB) and also challenged. ThisEB vaccine had shown great promise in field trials but is too expensiveto produce. After a prime and one boost, the heifers were estrussynchronized by prostaglandin injection, were in heat 2-3 days later,and were artificially inseminated, simultaneously receiving anintracervical chlamydial challenge of 3×10⁷ inclusion forming units. Theheifers were palpated for pregnancy at six weeks after insemination.This challenge was very high in order to maximize the difference betweenpositive and negative control animals. This was necessary because only asmall number of cows could be justified for this high-risk experiment.

[0267] Although the animal numbers are small, the results are quiteencouraging. As is seen in Table 1, three out of four animals becamepregnant in the positive control (non-challenged) group, 0/4 in thenegative control (non-vaccinated, challenged) group, 2/6 in the geneticimmunization group, and 1/4 in the elementary body vaccine group. Thegenetic vaccine of the pooled genes performed at least as well as the EBvaccine. Also relative to the inventor's interest in therapeuticvaccines, these cows were not sterile with respect to C. psittaci at thetime of the prime inoculation. The vaccination was in the face ofprevious exposure and low level C. psittaci infection, as determined bythe high titers of preinoculation antichlamydial antibodies, andoccasional positivity of Chlamydia omp1 PCRs from vaginal scrapings.

[0268] The next phase in developing a cow vaccine will be toexperimentally verify the effectiveness of particular groups of theprotective genes and then convert the codon usage of the C. psittacigenes to that of a mammal. This should increase the expression of theantigen in cows and increase the effectiveness of the vaccine. Theinventors will test different combinations of those genes which havebeen found to be individually protective, as well as combinations withCP4 #11. Both the original fragments and their full-length versions canbe tested, both as nucleic acid segments and proteins. Once thecombinations have been verified in mice or other small mammals, thosecombinations showing the most promise will be tested in cows. Afterimmunization, the cows will be challenged with C. psittaci, either bydirect challenge at insemination or infection by herd-mates. Directchallenge at insemination is a very severe and unnatural form ofchallenge. Therefore, even if protection is not demonstrated in the wakeof such challenge, this does not necessarily mean that no protection hasbeen conferred upon the cows.

[0269] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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[0374]

1 61 1 127 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 1 ctgcacctgg tccttcgcct gagaggtgca gatcttggatcctaagtaag taagcttgca 60 tgcctgcagg tcgactctag gtgactaata tctagaggatcgatcccggg tggcatccct 120 gtgaccc 127 2 15 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 2 gatctggatc ccgat15 3 11 DNA Artificial Sequence Description of Artificial SequenceSynthetic Primer 3 atcgggctcc a 11 4 19 DNA Artificial SequenceDescription of Artificial Sequence Synthetic Primer 4 ccgcaccctctctgattac 19 5 17 DNA Artificial Sequence Description of ArtificialSequence Synthetic Primer 5 ctggagtggc aacttcc 17 6 449 DNA ArtificialSequence Description of Artificial Sequence Synthetic Primer 6gaatgtattc gcacgcaaaa atacgctgaa gctttgcttc ctgtcacgac agcgatcaat 60tctggagtcg cgcctatcac cttcctccat gacctcactg ttttttatcg cgatgtactg 120ctaaacaaag atcagggaaa ttctcctcta tcggccatcg ccatgcacta ttccagtgaa 180tgtttattag aaatcattga tttccttggt gaagcggcca aacatctaca acaaactatt 240tttgaaaaaa catttttaga aacagtcatc atccatctta ttcggatatg ccaacgtccc 300tctttagaaa ctctgttttc tcaactgaaa acatccacgt ttgatacagt gagaaacgta 360ccccagcagc aagaaccctc gaaaccgagt atacaacctg aaaaacacta tcaagatcag 420agtttcttaa cttcaccttc tcccacgcc 449 7 149 PRT Chlamydia psittaci 7 GluCys Ile Arg Thr Gln Lys Tyr Ala Glu Ala Leu Leu Pro Val Thr 1 5 10 15Thr Ala Ile Asn Ser Gly Val Ala Pro Ile Thr Phe Leu His Asp Leu 20 25 30Thr Val Phe Tyr Arg Asp Val Leu Leu Asn Lys Asp Gln Gly Asn Ser 35 40 45Pro Leu Ser Ala Ile Ala Met His Tyr Ser Ser Glu Cys Leu Leu Glu 50 55 60Ile Ile Asp Phe Leu Gly Glu Ala Ala Lys His Leu Gln Gln Thr Ile 65 70 7580 Phe Glu Lys Thr Phe Leu Glu Thr Val Ile Ile His Leu Ile Arg Ile 85 9095 Cys Gln Arg Pro Ser Leu Glu Thr Leu Phe Ser Gln Leu Lys Thr Ser 100105 110 Thr Phe Asp Thr Val Arg Asn Val Pro Gln Gln Gln Glu Pro Ser Lys115 120 125 Pro Ser Ile Gln Pro Glu Lys His Tyr Gln Asp Gln Ser Phe LeuThr 130 135 140 Ser Pro Ser Pro Thr 145 8 1332 DNA Chlamydia psittaci 8atgacatcag caacatacca agtttcttct agaaaatacc gccctcaaac atttgccgaa 60atgctggggc aagatgccgt ggtcactgtt ttaaaaaatg ctttgcagtt tcaacgtgtc 120gcgcatgcgt atttattttc agggattcgc ggaacaggaa aaacaacttt agcaagaatc 180tttgcaaaag ccttaaactg taaagagctg actcctgaac atgaaccatg caaccagtgt 240tgtgtttgta aagaaatctc ttcaggaacc tccttagacg tgatcgaaat cgatggtgcc 300tcgcaccgag gtattgaaga tatccgtcaa atcaatgaaa ccgtgctctt tactcctgcc 360aaatcacaat ataaaatcta tatcatagat gaagtccata tgctgactaa ggaggcgttt 420aattccttac tcaaaacttt agaagagcct ccgagccatg taaaattctt cttagcgact 480acagaaaatt ataaaatacc cagcaccatt ttaagtcgtt gtcaaaaaat gcacctaaag 540agaattcctg agacaatgat tgtagataag ctagcatcca tatctcaagc aggtgggata 600gaaacctctc gagaagctct tcttcctatt gctagagcag cacagggaag cttacgcgat 660gctgaatctc tttatgatta tgtcataggg ttattcccta catctttatc cccagagttg 720gttgcagacg cattaggttt attatctcaa gacaccttag ctacattatc agaatgtatt 780cgcacgcaaa aatacgctga agctttgctt cctgtcacga cagcgatcaa ttctggagtc 840gcgcctatca ccttcctcca tgacctcact gttttttatc gcgatgtact gctaaacaaa 900gatcagggaa attctcctct atcggccatc gccatgcact attccagtga atgtttatta 960gaaatcattg atttccttgg tgaagcggcc aaacatctac aacaaactat ttttgaaaaa 1020acatttttag aaacagtcat catccatctt attcggatat gccaacgtcc ctctttagaa 1080actctgtttt ctcaactgaa aacatccacg tttgatacag tgagaaacgt accccagcag 1140caagaaccct cgaaaccgag tatacaacct gaaaaacact atcaagatca gagtttctta 1200acttcacctt ctcccacgcc aaaagttcag catcaaaaag aagcttcccc ttctttagtg 1260ggatcagcta ctatagatac gcttttacaa tttgctgttg ttgagttttc cggaatttta 1320accaaggagt aa 1332 9 443 PRT Chlamydia psittaci 9 Met Thr Ser Ala ThrTyr Gln Val Ser Ser Arg Lys Tyr Arg Pro Gln 1 5 10 15 Thr Phe Ala GluMet Leu Gly Gln Asp Ala Val Val Thr Val Leu Lys 20 25 30 Asn Ala Leu GlnPhe Gln Arg Val Ala His Ala Tyr Leu Phe Ser Gly 35 40 45 Ile Arg Gly ThrGly Lys Thr Thr Leu Ala Arg Ile Phe Ala Lys Ala 50 55 60 Leu Asn Cys LysGlu Leu Thr Pro Glu His Glu Pro Cys Asn Gln Cys 65 70 75 80 Cys Val CysLys Glu Ile Ser Ser Gly Thr Ser Leu Asp Val Ile Glu 85 90 95 Ile Asp GlyAla Ser His Arg Gly Ile Glu Asp Ile Arg Gln Ile Asn 100 105 110 Glu ThrVal Leu Phe Thr Pro Ala Lys Ser Gln Tyr Lys Ile Tyr Ile 115 120 125 IleAsp Glu Val His Met Leu Thr Lys Glu Ala Phe Asn Ser Leu Leu 130 135 140Lys Thr Leu Glu Glu Pro Pro Ser His Val Lys Phe Phe Leu Ala Thr 145 150155 160 Thr Glu Asn Tyr Lys Ile Pro Ser Thr Ile Leu Ser Arg Cys Gln Lys165 170 175 Met His Leu Lys Arg Ile Pro Glu Thr Met Ile Val Asp Lys LeuAla 180 185 190 Ser Ile Ser Gln Ala Gly Gly Ile Glu Thr Ser Arg Glu AlaLeu Leu 195 200 205 Pro Ile Ala Arg Ala Ala Gln Gly Ser Leu Arg Asp AlaGlu Ser Leu 210 215 220 Tyr Asp Tyr Val Ile Gly Leu Phe Pro Thr Ser LeuSer Pro Glu Leu 225 230 235 240 Val Ala Asp Ala Leu Gly Leu Leu Ser GlnAsp Thr Leu Ala Thr Leu 245 250 255 Ser Glu Cys Ile Arg Thr Gln Lys TyrAla Glu Ala Leu Leu Pro Val 260 265 270 Thr Thr Ala Ile Asn Ser Gly ValAla Pro Ile Thr Phe Leu His Asp 275 280 285 Leu Thr Val Phe Tyr Arg AspVal Leu Leu Asn Lys Asp Gln Gly Asn 290 295 300 Ser Pro Leu Ser Ala IleAla Met His Tyr Ser Ser Glu Cys Leu Leu 305 310 315 320 Glu Ile Ile AspPhe Leu Gly Glu Ala Ala Lys His Leu Gln Gln Thr 325 330 335 Ile Phe GluLys Thr Phe Leu Glu Thr Val Ile Ile His Leu Ile Arg 340 345 350 Ile CysGln Arg Pro Ser Leu Glu Thr Leu Phe Ser Gln Leu Lys Thr 355 360 365 SerThr Phe Asp Thr Val Arg Asn Val Pro Gln Gln Gln Glu Pro Ser 370 375 380Lys Pro Ser Ile Gln Pro Glu Lys His Tyr Gln Asp Gln Ser Phe Leu 385 390395 400 Thr Ser Pro Ser Pro Thr Pro Lys Val Gln His Gln Lys Glu Ala Ser405 410 415 Pro Ser Leu Val Gly Ser Ala Thr Ile Asp Thr Leu Leu Gln PheAla 420 425 430 Val Val Glu Phe Ser Gly Ile Leu Thr Lys Glu 435 440 10123 DNA Chlamydia psittaci 10 gagtttattc aagagtatga aagttctttaaatgaagtca ttaaaactat ggcagcatcc 60 atcgctatgg atgtaaccga cgtggttattgaggttggtt tatcccatgt gatcagtccc 120 gaa 123 11 41 PRT Chlamydiapsittaci 11 Glu Phe Ile Gln Glu Tyr Glu Ser Ser Leu Asn Glu Val Ile LysThr 1 5 10 15 Met Ala Ala Ser Ile Ala Met Asp Val Thr Asp Val Val IleGlu Val 20 25 30 Gly Leu Ser His Val Ile Ser Pro Glu 35 40 12 303 DNAChlamydia psittaci 12 atgacacaac cctatgtaac tagagaagac attatacttctggcgaagag ttcagctctg 60 gaattaagcg aagagtttat tcaagagtat gaaagttctttaaatgaagt cattaaaact 120 atggcagcat ccatcgctat ggatgtaacc gacgtggttattgaggttgg tttatcccat 180 gtgatcagtc ccgaagattt acgagaagat atcgttgcctcaagtttctc tcgtgaggag 240 tttctaacta atgtccctga atccttaggg ggattagtaaaagtacccac agtcattaag 300 tag 303 13 100 PRT Chlamydia psittaci 13 MetThr Gln Pro Tyr Val Thr Arg Glu Asp Ile Ile Leu Leu Ala Lys 1 5 10 15Ser Ser Ala Leu Glu Leu Ser Glu Glu Phe Ile Gln Glu Tyr Glu Ser 20 25 30Ser Leu Asn Glu Val Ile Lys Thr Met Ala Ala Ser Ile Ala Met Asp 35 40 45Val Thr Asp Val Val Ile Glu Val Gly Leu Ser His Val Ile Ser Pro 50 55 60Glu Asp Leu Arg Glu Asp Ile Val Ala Ser Ser Phe Ser Arg Glu Glu 65 70 7580 Phe Leu Thr Asn Val Pro Glu Ser Leu Gly Gly Leu Val Lys Val Pro 85 9095 Thr Val Ile Lys 100 14 514 DNA Chlamydia psittaci 14 gaaaagtgtgatgtgattgc gatgcctgta tgctcatgcc cagcattcgc cgatggcgaa 60 atccttgatcctacctctct atatctccag gatatctata ccgtggctat gaatttagcc 120 tacctcccagctatcgccgt tccttcaggg ttttctcgag aagggctgcc tctaggattc 180 caggtgattggacaaaaggg taaagatcaa caggtgtgcc aggtaggcta tagcttccaa 240 gaacattcaggaattaagaa tttataccct aaaggatgta acaaacttgt tgatggagag 300 gtgaaataatgagcgacgtt tatgctgatt gggaatccgt cataggtctt gaagtccacg 360 tagaattaaacacaaaatct aaattgttca gttgtgcacg caaccgtttt ggagacgaac 420 ctaatacaaacatctctcct gtatgcaccg gcatgccggg gtcactgcca gtactgaata 480 aagaagcagtgagaaaggct gttttatttg gttg 514 15 102 PRT Chlamydia psittaci 15 Glu LysCys Asp Val Ile Ala Met Pro Val Cys Ser Cys Pro Ala Phe 1 5 10 15 AlaAsp Gly Glu Ile Leu Asp Pro Thr Ser Leu Tyr Leu Gln Asp Ile 20 25 30 TyrThr Val Ala Met Asn Leu Ala Tyr Leu Pro Ala Ile Ala Val Pro 35 40 45 SerGly Phe Ser Arg Glu Gly Leu Pro Leu Gly Phe Gln Val Ile Gly 50 55 60 GlnLys Gly Lys Asp Gln Gln Val Cys Gln Val Gly Tyr Ser Phe Gln 65 70 75 80Glu His Ser Gly Ile Lys Asn Leu Tyr Pro Lys Gly Cys Asn Lys Leu 85 90 95Val Asp Gly Glu Val Lys 100 16 1476 DNA Chlamydia psittaci 16 atgtatcagaagagtgcctt agagttaaga aatgccgtag tgagtggaga gtcttcagct 60 acagcaatagcaaagtattt ttataataga ataaaaacag aagacaatca gataggagct 120 tttctttctctttgtgaaga aagagcttat gagaaagcag ctatcataga tgcgaaagtg 180 gcgcgaggagaacctttggg gaaactcgca ggtgtcccca tcgggataaa agataatatt 240 catattcggggtttgcgcac cacttgtgct tctaaaatgt tagaaaatta tatagcgcct 300 tttgatgctacagtcgtcga acggatagaa gctgaagatg gggtcatttt aggcaaactc 360 aatatggatgagtttgctat gggatcgaca acgcagtatt ctgctttcca tcctacgaaa 420 aatccttggggtttatcctg tgtgccagga ggatcttcag ggggatccgc cgccgcagtt 480 tctgcaagattttgtcctat agcgttaggt tcggataccg gtggatctat acgtcagcca 540 gcagcattttgtggagttgt ggggtttaag ccctcctatg gagccgtctc ccgttacggt 600 ttagtcgcttttgggtcttc attagatcag ataggccctt taacaacagt tgtcgaagat 660 gtcgccttagctatggatgt attcgcaggt aaggatgata gagatgcaac ttctcagaag 720 ttttttacaggatctttcca agaggccttg tctttagacg ttccgagttt gatcggcgtg 780 cctatgggatttttagacgg tttacgtgat gatgttaaag agaatttctt tgcctcttta 840 agtattttggaacgtcaggg tagccgcatt gttgaagtgg atcttaacat cttagatcac 900 gctgtctctgtttactacat tgtcgcttct gcagaagccg caacaaatct tgcaagattt 960 gatggtattcgttacggcta tcgttctcca gaagcgcata gtatagaaga tatttatacg 1020 atctcccgcgtacaaggctt cggtaaggaa gtcatgcgta ggattctttt aggtaactat 1080 gtgttatccactgagcgcca aaatgtctat tataagaaag gctccgcaat tcgagcaaaa 1140 atcattcaagcttttcaaaa agcttatgaa aagtgtgatg tgattgcgat gcctgtatgc 1200 tcatgcccagcattcgccga tggcgaaatc cttgatccta cctctctata tctccaggat 1260 atctataccgtggctatgaa tttagcctac ctcccagcta tcgccgttcc ttcagggttt 1320 tctcgagaagggctgcctct aggattccag gtgattggac aaaagggtaa agatcaacag 1380 gtgtgccaggtaggctatag cttccaagaa cattcaggaa ttaagaattt ataccctaaa 1440 ggatgtaacaaacttgttga tggagaggtg aaataa 1476 17 491 PRT Chlamydia psittaci 17 MetTyr Gln Lys Ser Ala Leu Glu Leu Arg Asn Ala Val Val Ser Gly 1 5 10 15Glu Ser Ser Ala Thr Ala Ile Ala Lys Tyr Phe Tyr Asn Arg Ile Lys 20 25 30Thr Glu Asp Asn Gln Ile Gly Ala Phe Leu Ser Leu Cys Glu Glu Arg 35 40 45Ala Tyr Glu Lys Ala Ala Ile Ile Asp Ala Lys Val Ala Arg Gly Glu 50 55 60Pro Leu Gly Lys Leu Ala Gly Val Pro Ile Gly Ile Lys Asp Asn Ile 65 70 7580 His Ile Arg Gly Leu Arg Thr Thr Cys Ala Ser Lys Met Leu Glu Asn 85 9095 Tyr Ile Ala Pro Phe Asp Ala Thr Val Val Glu Arg Ile Glu Ala Glu 100105 110 Asp Gly Val Ile Leu Gly Lys Leu Asn Met Asp Glu Phe Ala Met Gly115 120 125 Ser Thr Thr Gln Tyr Ser Ala Phe His Pro Thr Lys Asn Pro TrpGly 130 135 140 Leu Ser Cys Val Pro Gly Gly Ser Ser Gly Gly Ser Ala AlaAla Val 145 150 155 160 Ser Ala Arg Phe Cys Pro Ile Ala Leu Gly Ser AspThr Gly Gly Ser 165 170 175 Ile Arg Gln Pro Ala Ala Phe Cys Gly Val ValGly Phe Lys Pro Ser 180 185 190 Tyr Gly Ala Val Ser Arg Tyr Gly Leu ValAla Phe Gly Ser Ser Leu 195 200 205 Asp Gln Ile Gly Pro Leu Thr Thr ValVal Glu Asp Val Ala Leu Ala 210 215 220 Met Asp Val Phe Ala Gly Lys AspAsp Arg Asp Ala Thr Ser Gln Lys 225 230 235 240 Phe Phe Thr Gly Ser PheGln Glu Ala Leu Ser Leu Asp Val Pro Ser 245 250 255 Leu Ile Gly Val ProMet Gly Phe Leu Asp Gly Leu Arg Asp Asp Val 260 265 270 Lys Glu Asn PhePhe Ala Ser Leu Ser Ile Leu Glu Arg Gln Gly Ser 275 280 285 Arg Ile ValGlu Val Asp Leu Asn Ile Leu Asp His Ala Val Ser Val 290 295 300 Tyr TyrIle Val Ala Ser Ala Glu Ala Ala Thr Asn Leu Ala Arg Phe 305 310 315 320Asp Gly Ile Arg Tyr Gly Tyr Arg Ser Pro Glu Ala His Ser Ile Glu 325 330335 Asp Ile Tyr Thr Ile Ser Arg Val Gln Gly Phe Gly Lys Glu Val Met 340345 350 Arg Arg Ile Leu Leu Gly Asn Tyr Val Leu Ser Thr Glu Arg Gln Asn355 360 365 Val Tyr Tyr Lys Lys Gly Ser Ala Ile Arg Ala Lys Ile Ile GlnAla 370 375 380 Phe Gln Lys Ala Tyr Glu Lys Cys Asp Val Ile Ala Met ProVal Cys 385 390 395 400 Ser Cys Pro Ala Phe Ala Asp Gly Glu Ile Leu AspPro Thr Ser Leu 405 410 415 Tyr Leu Gln Asp Ile Tyr Thr Val Ala Met AsnLeu Ala Tyr Leu Pro 420 425 430 Ala Ile Ala Val Pro Ser Gly Phe Ser ArgGlu Gly Leu Pro Leu Gly 435 440 445 Phe Gln Val Ile Gly Gln Lys Gly LysAsp Gln Gln Val Cys Gln Val 450 455 460 Gly Tyr Ser Phe Gln Glu His SerGly Ile Lys Asn Leu Tyr Pro Lys 465 470 475 480 Gly Cys Asn Lys Leu ValAsp Gly Glu Val Lys 485 490 18 1464 DNA Chlamydia psittaci 18 atgagcgacgtttatgctga ttgggaatcc gtcataggtc ttgaagtcca cgtagaatta 60 aacacaaaatctaaattgtt cagttgtgca cgcaaccgtt ttggagacga acctaataca 120 aacatctctcctgtatgcac cggcatgccg gggtcactgc cagtactgaa taaagaagca 180 gtgagaaaggctgttttatt tggttgtgct gttgaaggcg aagtagcttt gctcagccgt 240 tttgatagaaagtcctattt ttatcccgat agcccaagga attttcaaat tacccaattc 300 gaacatcctattgtgcgagg aggacatata aaagctatcg ttcacggtga ggaacgtcat 360 tttgaactggctcaagcgca tatcgaagat gatgccggta tgctaaaaca tttcggagaa 420 tttgctggagtagattataa ccgcgctggt gtacctttaa tagagattgt gtctaagccg 480 tgcatgttttgtgctgatga tgctgttgct tatgccacag ctttggtatc cttattagac 540 tacataggcatttctgactg taatatggaa gaaggctcgg tacgctttga tgtaaacata 600 tccgtacgtcctaaaggtag cgaagaacta cgcaataaag tagaaattaa aaatatgaac 660 tcctttgcttttatggccca agctctagaa gccgagcgtt gtcgtcagat cgatgcatat 720 ttagacaatccaaatgcaga ccccaaaact gttattccag gagcgacata ccgttgggat 780 cctgaaaagaaaaaaacagt gttgatgcgt cttaaggaac gagctgaaga ttacaagtat 840 ttcatagagcctgatctccc agtattgcaa ttaacagaag catatattga tgaaattcgt 900 catacgcttcccgagctccc tttcaacaaa taccaaaggt atttgcacga atatgctctt 960 gccgaagacatcgctgccat tttaattagc gataagcata gtgcgcactt ctttgaatta 1020 gccgctcaggaatgtaaaaa ctacagagcc ctttctaatt ggttaactgt tgagtttgcc 1080 ggacgttgtaaactcaaggg taagaatctc gctttctcag gtatcctgcc cagtagtgta 1140 gctcagcttgtgaattttat tgatcaaggc gtgattaccg gaaagatcgc taaggatatc 1200 gcagacatgatgatggaatc tcctgaaaag agtcctgaga ctatcctcaa agaaaatcct 1260 gaaatgttgcccatgacaga tgaaagtgcg ttggtggcga tcatttccga ggtgattacc 1320 gcaaatccgcagtctgtcgt agactacaaa agtggtaaga ccaaggcgtt aggattttta 1380 gttgggcaaattatgaaacg tacccagggc aaggcccctc caaatagggt aaatgaactt 1440 ttgcttgtggaattaagtaa ataa 1464 19 487 PRT Chlamydia psittaci 19 Met Ser Asp ValTyr Ala Asp Trp Glu Ser Val Ile Gly Leu Glu Val 1 5 10 15 His Val GluLeu Asn Thr Lys Ser Lys Leu Phe Ser Cys Ala Arg Asn 20 25 30 Arg Phe GlyAsp Glu Pro Asn Thr Asn Ile Ser Pro Val Cys Thr Gly 35 40 45 Met Pro GlySer Leu Pro Val Leu Asn Lys Glu Ala Val Arg Lys Ala 50 55 60 Val Leu PheGly Cys Ala Val Glu Gly Glu Val Ala Leu Leu Ser Arg 65 70 75 80 Phe AspArg Lys Ser Tyr Phe Tyr Pro Asp Ser Pro Arg Asn Phe Gln 85 90 95 Ile ThrGln Phe Glu His Pro Ile Val Arg Gly Gly His Ile Lys Ala 100 105 110 IleVal His Gly Glu Glu Arg His Phe Glu Leu Ala Gln Ala His Ile 115 120 125Glu Asp Asp Ala Gly Met Leu Lys His Phe Gly Glu Phe Ala Gly Val 130 135140 Asp Tyr Asn Arg Ala Gly Val Pro Leu Ile Glu Ile Val Ser Lys Pro 145150 155 160 Cys Met Phe Cys Ala Asp Asp Ala Val Ala Tyr Ala Thr Ala LeuVal 165 170 175 Ser Leu Leu Asp Tyr Ile Gly Ile Ser Asp Cys Asn Met GluGlu Gly 180 185 190 Ser Val Arg Phe Asp Val Asn Ile Ser Val Arg Pro LysGly Ser Glu 195 200 205 Glu Leu Arg Asn Lys Val Glu Ile Lys Asn Met AsnSer Phe Ala Phe 210 215 220 Met Ala Gln Ala Leu Glu Ala Glu Arg Cys ArgGln Ile Asp Ala Tyr 225 230 235 240 Leu Asp Asn Pro Asn Ala Asp Pro LysThr Val Ile Pro Gly Ala Thr 245 250 255 Tyr Arg Trp Asp Pro Glu Lys LysLys Thr Val Leu Met Arg Leu Lys 260 265 270 Glu Arg Ala Glu Asp Tyr LysTyr Phe Ile Glu Pro Asp Leu Pro Val 275 280 285 Leu Gln Leu Thr Glu AlaTyr Ile Asp Glu Ile Arg His Thr Leu Pro 290 295 300 Glu Leu Pro Phe AsnLys Tyr Gln Arg Tyr Leu His Glu Tyr Ala Leu 305 310 315 320 Ala Glu AspIle Ala Ala Ile Leu Ile Ser Asp Lys His Ser Ala His 325 330 335 Phe PheGlu Leu Ala Ala Gln Glu Cys Lys Asn Tyr Arg Ala Leu Ser 340 345 350 AsnTrp Leu Thr Val Glu Phe Ala Gly Arg Cys Lys Leu Lys Gly Lys 355 360 365Asn Leu Ala Phe Ser Gly Ile Leu Pro Ser Ser Val Ala Gln Leu Val 370 375380 Asn Phe Ile Asp Gln Gly Val Ile Thr Gly Lys Ile Ala Lys Asp Ile 385390 395 400 Ala Asp Met Met Met Glu Ser Pro Glu Lys Ser Pro Glu Thr IleLeu 405 410 415 Lys Glu Asn Pro Glu Met Leu Pro Met Thr Asp Glu Ser AlaLeu Val 420 425 430 Ala Ile Ile Ser Glu Val Ile Thr Ala Asn Pro Gln SerVal Val Asp 435 440 445 Tyr Lys Ser Gly Lys Thr Lys Ala Leu Gly Phe LeuVal Gly Gln Ile 450 455 460 Met Lys Arg Thr Gln Gly Lys Ala Pro Pro AsnArg Val Asn Glu Leu 465 470 475 480 Leu Leu Val Glu Leu Ser Lys 485 20379 DNA Chlamydia psittaci 20 tatttagtgt cgaaaaacaa cgccaacatttacgcaggtt ctctctatta tcagcatatc 60 tcctattgga gcgcttggca gaatctgctacaaaacacta tcggtgcaga agctccgtta 120 gtccttaacg cacagttaac ttattgtcatgcttcaaacg acatgaaaac caacatgacg 180 actacttacg ctcctcgtaa aacaacgtatgcagaaatca agggtgattg gggtaacgat 240 tgtttcggag tcgagcttgg tgcaactgtgcctatccaaa cagaatcttc tctcctattc 300 gatatgtact cacctttcct gaagtttcaacttgtgcata cgcaccaaga tgactttaag 360 gaaaacaata gcgatcagg 379 21 126 PRTChlamydia psittaci 21 Tyr Leu Val Ser Lys Asn Asn Ala Asn Ile Tyr AlaGly Ser Leu Tyr 1 5 10 15 Tyr Gln His Ile Ser Tyr Trp Ser Ala Trp GlnAsn Leu Leu Gln Asn 20 25 30 Thr Ile Gly Ala Glu Ala Pro Leu Val Leu AsnAla Gln Leu Thr Tyr 35 40 45 Cys His Ala Ser Asn Asp Met Lys Thr Asn MetThr Thr Thr Tyr Ala 50 55 60 Pro Arg Lys Thr Thr Tyr Ala Glu Ile Lys GlyAsp Trp Gly Asn Asp 65 70 75 80 Cys Phe Gly Val Glu Leu Gly Ala Thr ValPro Ile Gln Thr Glu Ser 85 90 95 Ser Leu Leu Phe Asp Met Tyr Ser Pro PheLeu Lys Phe Gln Leu Val 100 105 110 His Thr His Gln Asp Asp Phe Lys GluAsn Asn Ser Asp Gln 115 120 125 22 2520 DNA Chlamydia psittaci 22atgaaacatc cagtctactg gttcttaata tcctcgagcc tatttgcctc gaattctttg 60agcttcgcta acgacgctca aacagcctta actccctccg atagctataa tggaaatgtg 120acctctgagg agttccaggt aaaagaaact tcatcaggaa caacgtatac ttgtgaaggc 180aatgtgtgta tctcctttgc agggaaagat tcaggtctaa agaaaagttg tttctcagct 240actgataacc ttaccttcct aggaaacggg tatactcttt gctttgataa tattactact 300acagctagta accccggagc cattaatgtt caaggtcaag gaaaaacctt aggcatctca 360ggattttctt tattttcatg tgcttattgt cctccaggca caactggtta cggagctata 420cagactaaag gcaacacaac tttaaaagat aactctagtc ttgtcttcca taaaaactgc 480tcaacagcag aaggtggggc tatccagtgt aaaggaagca gtgatgctga attaaaaata 540gaaaataatc agaatctggt tttctcagaa aactcctcca cttcaaaagg cggggctatt 600tatgctgata aactcaccat tgtctcaggt gggcctacat tattttctaa caactctgta 660tccaacggtt catcccctaa aggcggagct attagcataa aagattcaag tggtgaatgt 720agcctaaccg ctgatctcgg agatattacc ttcgatggga acaaaatcat caaaactagt 780ggtggaagtt ctacagtaac aagaaattcc atagatctcg gcacagggaa atttacaaag 840ctacgtgcta aagacggctt cggaattttc ttctatgacc ctattactgg gggaggatct 900gatgaactaa acattaataa aaaagaaact gttgattata caggaaagat cgtcttctca 960ggtgaaaaat tatccgatga agaaaaagca cgagcggaaa acctagcttc tactttcaac 1020caacccatca cattatcagc aggatctctt gtacttaaag atggtgtatc tgtaaccgca 1080aaacaagtaa cgcaggaagc gggatctacc gttgtcatgg atctagggac cacattacag 1140acgccttctt caggtggaga aaccatcacc ctaactaatc tagatattaa catcgcctcg 1200ttgggggggg gggggggtac ctctcctgct aaactcgcaa caaatacagc aagtcaagct 1260ataactatta acgctgtcaa tctagtcgat gctgatggca atgcttatga agatcctatt 1320cttgctacgt ctaaaccttt cacagcaata gtagctacaa ctaacgctag tacagtcaca 1380cagcctacag ataatctaac aaattatgtc cctcctactc attacggtta ccaaggaaat 1440tggacagtaa cttgggacac cgaaacagct acaaaaacag ccactctaac ttgggaacaa 1500actggctact cccctaaccc agaacgtcaa ggacctttag tcccgaatac tctttggggt 1560gcattctctg acctcagagc tatacaaaac ttaatggata ttagcgtcaa tggcgctgac 1620taccatagag gtttttgggt atccggtcta gctaacttct tacacaaaag tggctctgat 1680actaaacgca agttccgtca caatagcgcc ggatacgctt taggcgtcta cgcaaaaact 1740ccttctgatg atattttcag tgcggctttc tgccaactct tcggaaagga caaagactat 1800ttagtgtcga aaaacaacgc caacatttac gcaggttctc tctattatca gcatatctcc 1860tattggagcg cttggcagaa tctgctacaa aacactatcg gtgcagaagc tccgttagtc 1920cttaacgcac agttaactta ttgtcatgct tcaaacgaca tgaaaaccaa catgacgact 1980acttacgctc ctcgtaaaac aacgtatgca gaaatcaagg gtgattgggg taacgattgt 2040ttcggagtcg agcttggtgc aactgtgcct atccaaacag aatcttctct cctattcgat 2100atgtactcac ctttcctgaa gtttcaactt gtgcatacgc accaagatga ctttaaggaa 2160aacaatagcg atcagggaag atacttcgaa agcagcaatc tcaccaacct ttctctgcct 2220atcggcatca agtttgagag atttgctaac aacgatacag cttcttatca tgtcactgct 2280gcttattctc ctgatatcgt aagaagtaac cctgactgta ctacttctct gttagtaagc 2340cccgactctg ctgtctgggt aacgaaagcc aacaaccttg cgcgaagcgc cttcatgcta 2400caagcaggaa actacttgtc tttaagtcac aacatagaaa tcttcagcca gttcggtttc 2460gagctcaggg gatcttcacg aacctataac gtagatctcg gatcgaagat ccagttctaa 252023 839 PRT Chlamydia psittaci 23 Met Lys His Pro Val Tyr Trp Phe Leu IleSer Ser Ser Leu Phe Ala 1 5 10 15 Ser Asn Ser Leu Ser Phe Ala Asn AspAla Gln Thr Ala Leu Thr Pro 20 25 30 Ser Asp Ser Tyr Asn Gly Asn Val ThrSer Glu Glu Phe Gln Val Lys 35 40 45 Glu Thr Ser Ser Gly Thr Thr Tyr ThrCys Glu Gly Asn Val Cys Ile 50 55 60 Ser Phe Ala Gly Lys Asp Ser Gly LeuLys Lys Ser Cys Phe Ser Ala 65 70 75 80 Thr Asp Asn Leu Thr Phe Leu GlyAsn Gly Tyr Thr Leu Cys Phe Asp 85 90 95 Asn Ile Thr Thr Thr Ala Ser AsnPro Gly Ala Ile Asn Val Gln Gly 100 105 110 Gln Gly Lys Thr Leu Gly IleSer Gly Phe Ser Leu Phe Ser Cys Ala 115 120 125 Tyr Cys Pro Pro Gly ThrThr Gly Tyr Gly Ala Ile Gln Thr Lys Gly 130 135 140 Asn Thr Thr Leu LysAsp Asn Ser Ser Leu Val Phe His Lys Asn Cys 145 150 155 160 Ser Thr AlaGlu Gly Gly Ala Ile Gln Cys Lys Gly Ser Ser Asp Ala 165 170 175 Glu LeuLys Ile Glu Asn Asn Gln Asn Leu Val Phe Ser Glu Asn Ser 180 185 190 SerThr Ser Lys Gly Gly Ala Ile Tyr Ala Asp Lys Leu Thr Ile Val 195 200 205Ser Gly Gly Pro Thr Leu Phe Ser Asn Asn Ser Val Ser Asn Gly Ser 210 215220 Ser Pro Lys Gly Gly Ala Ile Ser Ile Lys Asp Ser Ser Gly Glu Cys 225230 235 240 Ser Leu Thr Ala Asp Leu Gly Asp Ile Thr Phe Asp Gly Asn LysIle 245 250 255 Ile Lys Thr Ser Gly Gly Ser Ser Thr Val Thr Arg Asn SerIle Asp 260 265 270 Leu Gly Thr Gly Lys Phe Thr Lys Leu Arg Ala Lys AspGly Phe Gly 275 280 285 Ile Phe Phe Tyr Asp Pro Ile Thr Gly Gly Gly SerAsp Glu Leu Asn 290 295 300 Ile Asn Lys Lys Glu Thr Val Asp Tyr Thr GlyLys Ile Val Phe Ser 305 310 315 320 Gly Glu Lys Leu Ser Asp Glu Glu LysAla Arg Ala Glu Asn Leu Ala 325 330 335 Ser Thr Phe Asn Gln Pro Ile ThrLeu Ser Ala Gly Ser Leu Val Leu 340 345 350 Lys Asp Gly Val Ser Val ThrAla Lys Gln Val Thr Gln Glu Ala Gly 355 360 365 Ser Thr Val Val Met AspLeu Gly Thr Thr Leu Gln Thr Pro Ser Ser 370 375 380 Gly Gly Glu Thr IleThr Leu Thr Asn Leu Asp Ile Asn Ile Ala Ser 385 390 395 400 Leu Gly GlyGly Gly Gly Thr Ser Pro Ala Lys Leu Ala Thr Asn Thr 405 410 415 Ala SerGln Ala Ile Thr Ile Asn Ala Val Asn Leu Val Asp Ala Asp 420 425 430 GlyAsn Ala Tyr Glu Asp Pro Ile Leu Ala Thr Ser Lys Pro Phe Thr 435 440 445Ala Ile Val Ala Thr Thr Asn Ala Ser Thr Val Thr Gln Pro Thr Asp 450 455460 Asn Leu Thr Asn Tyr Val Pro Pro Thr His Tyr Gly Tyr Gln Gly Asn 465470 475 480 Trp Thr Val Thr Trp Asp Thr Glu Thr Ala Thr Lys Thr Ala ThrLeu 485 490 495 Thr Trp Glu Gln Thr Gly Tyr Ser Pro Asn Pro Glu Arg GlnGly Pro 500 505 510 Leu Val Pro Asn Thr Leu Trp Gly Ala Phe Ser Asp LeuArg Ala Ile 515 520 525 Gln Asn Leu Met Asp Ile Ser Val Asn Gly Ala AspTyr His Arg Gly 530 535 540 Phe Trp Val Ser Gly Leu Ala Asn Phe Leu HisLys Ser Gly Ser Asp 545 550 555 560 Thr Lys Arg Lys Phe Arg His Asn SerAla Gly Tyr Ala Leu Gly Val 565 570 575 Tyr Ala Lys Thr Pro Ser Asp AspIle Phe Ser Ala Ala Phe Cys Gln 580 585 590 Leu Phe Gly Lys Asp Lys AspTyr Leu Val Ser Lys Asn Asn Ala Asn 595 600 605 Ile Tyr Ala Gly Ser LeuTyr Tyr Gln His Ile Ser Tyr Trp Ser Ala 610 615 620 Trp Gln Asn Leu LeuGln Asn Thr Ile Gly Ala Glu Ala Pro Leu Val 625 630 635 640 Leu Asn AlaGln Leu Thr Tyr Cys His Ala Ser Asn Asp Met Lys Thr 645 650 655 Asn MetThr Thr Thr Tyr Ala Pro Arg Lys Thr Thr Tyr Ala Glu Ile 660 665 670 LysGly Asp Trp Gly Asn Asp Cys Phe Gly Val Glu Leu Gly Ala Thr 675 680 685Val Pro Ile Gln Thr Glu Ser Ser Leu Leu Phe Asp Met Tyr Ser Pro 690 695700 Phe Leu Lys Phe Gln Leu Val His Thr His Gln Asp Asp Phe Lys Glu 705710 715 720 Asn Asn Ser Asp Gln Gly Arg Tyr Phe Glu Ser Ser Asn Leu ThrAsn 725 730 735 Leu Ser Leu Pro Ile Gly Ile Lys Phe Glu Arg Phe Ala AsnAsn Asp 740 745 750 Thr Ala Ser Tyr His Val Thr Ala Ala Tyr Ser Pro AspIle Val Arg 755 760 765 Ser Asn Pro Asp Cys Thr Thr Ser Leu Leu Val SerPro Asp Ser Ala 770 775 780 Val Trp Val Thr Lys Ala Asn Asn Leu Ala ArgSer Ala Phe Met Leu 785 790 795 800 Gln Ala Gly Asn Tyr Leu Ser Leu SerHis Asn Ile Glu Ile Phe Ser 805 810 815 Gln Phe Gly Phe Glu Leu Arg GlySer Ser Arg Thr Tyr Asn Val Asp 820 825 830 Leu Gly Ser Lys Ile Gln Phe835 24 1039 DNA Chlamydia psittaci 24 aaacgttttc atattaatgg ggttcctgaatggtctttat ctacgcctta ttctcttgct 60 atggggtata atatcttggc tacgggagtgcagatggtta aagcctatgc cattcttgcc 120 aacggtggtt atgatgtgcg ccctaccttgataaaaaaaa tagtcactac ttctggaaaa 180 gagtacgtgt tgcatcctca agttcgtggagaaagaattc tttctcagga cattgtggat 240 gaggtattga aagctacgcg ttttactacctatcctggag gaacgggatt tcgggctgcg 300 cctaaaaagc attccagtgc agggaaaacaggaacaacag aaaagctagt tcatggaaag 360 tatgataagc atcggcatat ttcttcatttataggtatca cgccgatata cccttcggca 420 ggggggagtg ttcctttggt catgcttgtctctatcagtt atacgaccga caacggtagt 480 caagtgtacg tcgttcaatt gcgacatgagggtatcgaaa tctgtcgtca attcgtccat 540 gttaacctaa ttgtgtggtc attatcgctttctttatact acttaccgta gttcctacgg 600 atactagcaa aaagttctgc tctttgcgttgctctttgaa cagcatactg tacttttaaa 660 aagtctgcta aattttcccg ttctccattcctatctgaga agtagagaag ggctctattt 720 aacacttctt ctccagaaga cacccaattgaccatcttac gggcaacgga ctcgtgttct 780 tcttcttttt tggtttgtaa gttttgttgcgtatgctcag ctatatcatt cagatcacca 840 ttgattaaat caatgatcac actgacagcttcaaaatgtt cttgcgatag tttattttga 900 tcttgttgta gagtggattg tgcatcccataaacgctctt ttagattgtt tatttgctct 960 ttcagctctt ccgaatctaa cgcctcttccagttcaggat cgataatgtt agagtttctg 1020 tcttgcatca tcgccatag 1039 25 196PRT Chlamydia psittaci 25 Lys Arg Phe His Ile Asn Gly Val Pro Glu TrpSer Leu Ser Thr Pro 1 5 10 15 Tyr Ser Leu Ala Met Gly Tyr Asn Ile LeuAla Thr Gly Val Gln Met 20 25 30 Val Lys Ala Tyr Ala Ile Leu Ala Asn GlyGly Tyr Asp Val Arg Pro 35 40 45 Thr Leu Ile Lys Lys Ile Val Thr Thr SerGly Lys Glu Tyr Val Leu 50 55 60 His Pro Gln Val Arg Gly Glu Arg Ile LeuSer Gln Asp Ile Val Asp 65 70 75 80 Glu Val Leu Lys Ala Thr Arg Phe ThrThr Tyr Pro Gly Gly Thr Gly 85 90 95 Phe Arg Ala Ala Pro Lys Lys His SerSer Ala Gly Lys Thr Gly Thr 100 105 110 Thr Glu Lys Leu Val His Gly LysTyr Asp Lys His Arg His Ile Ser 115 120 125 Ser Phe Ile Gly Ile Thr ProIle Tyr Pro Ser Ala Gly Gly Ser Val 130 135 140 Pro Leu Val Met Leu ValSer Ile Ser Tyr Thr Thr Asp Asn Gly Ser 145 150 155 160 Gln Val Tyr ValVal Gln Leu Arg His Glu Gly Ile Glu Ile Cys Arg 165 170 175 Gln Phe ValHis Val Asn Leu Ile Val Trp Ser Leu Ser Leu Ser Leu 180 185 190 Tyr TyrLeu Pro 195 26 1950 DNA Chlamydia psittaci 26 atgaatcacc gtaaatgcttaaccatgatt acctatggag ttctgctctc ctattctttc 60 ctgatcatac ggtattataaaattcagatt tgtgaggaga aacgttgggc agcagaagct 120 ttaggacaac atgaatttcgagtaaaggac ccttttcgta gggggacgtt tttttctcag 180 atgaatttac gtaagggagattcagagcaa cgacaagctc tggccgtgga cattacgaaa 240 tttcatcttt gtttagatgctgtagctgtt cctgaagaac accgtgatgt gattgctaag 300 aaagttttta gtctcattggagaaggtgat tatgacaaac tccgtgcgga gtttgataaa 360 aaatctcgct atcgaaagttatttctttgg ttagatcgtg cggatcatga ccgcatcctg 420 tcttggtggc gggggtacgcagcaaaatct aaaataccct cgaatgcttt gtttttcatg 480 accgactatc aaagatcttatccctttggc aaacttttag gccaagttct acatactctg 540 agagaagtca aggatgagaaaacaggcaaa gctttcccta caggaggttt agaagcctat 600 tttaaccacg tccttgaaggagagccagga gaacggaaat tcctacgttc tcctttaaat 660 cgtttagatc tagataaagtcacaaagatt cctagggatg gttcggatat ttatctcaca 720 gtcaatccct gtatacagactatagcggaa gaggaattag aaaaaggggt aaaggaagcc 780 aaagctaaag gtgggcgtctaattttaatg aatgcttata caggcgagat tcttgcttta 840 gcacagtatc ctttctttaatccttcggaa tacaaggaat ttttcaatga taaggaaaaa 900 atagagcaca caaaagtaacatcagtcagt gatgtgtttg aacccggctc tatcatgaaa 960 cctctgactc tggctatagcgttgctggcc aacgaagaga tggtgaaaag atcaggaaag 1020 cccttatttg atcctaatgaacctatagat gtaacccgca ggattttccc aggaagaaag 1080 caatttccgc ttaaggatatctcatcgaat cggcgtttaa atatgtacat ggcgattcaa 1140 aagtcttcga acgtttatgtagcgcaactt gctgatctta tagtgcaaca tctagggaac 1200 cactggtatg aagacaagttattgttatta ggatttggta aaaagacggg gatagaattg 1260 ccaggggaag cgtcaggattggtaccttca cctaaacgtt ttcatattaa tggggttcct 1320 gaatggtctt tatctacgccttattctctt gctatggggt ataatatctt ggctacggga 1380 gtgcagatgg ttaaagcctatgccattctt gccaacggtg gttatgatgt gcgccctacc 1440 ttgataaaaa aaatagtcactacttctgga aaagagtacg tgttgcatcc tcaagttcgt 1500 ggagaaagaa ttctttctcaggacattgtg gatgaggtat tgaaagctac gcgttttact 1560 acctatcctg gaggaacgggatttcgggct gcgcctaaaa agcattccag tgcagggaaa 1620 acaggaacaa cagaaaagctagttcatgga aagtatgata agcatcggca tatttcttca 1680 tttataggta tcacgccgatatacccttcg gcagggggga gtgttccttt ggtcatgctt 1740 gtctctatag atgatcctgatcattgtgtt cgcgaggatg gaacaaagaa ctatatggga 1800 ggccgatgtg ccgcccctgtatttggcaga gttgcggatc gtgttttatc ttatctagga 1860 gttcccgaag ataaagaaaaatacagttat cagagtgagg tggctgctat gaaagctttg 1920 tatgaggaat ggaatcgttcggggaaataa 1950 27 649 PRT Chlamydia psittaci 27 Met Asn His Arg Lys CysLeu Thr Met Ile Thr Tyr Gly Val Leu Leu 1 5 10 15 Ser Tyr Ser Phe LeuIle Ile Arg Tyr Tyr Lys Ile Gln Ile Cys Glu 20 25 30 Glu Lys Arg Trp AlaAla Glu Ala Leu Gly Gln His Glu Phe Arg Val 35 40 45 Lys Asp Pro Phe ArgArg Gly Thr Phe Phe Ser Gln Met Asn Leu Arg 50 55 60 Lys Gly Asp Ser GluGln Arg Gln Ala Leu Ala Val Asp Ile Thr Lys 65 70 75 80 Phe His Leu CysLeu Asp Ala Val Ala Val Pro Glu Glu His Arg Asp 85 90 95 Val Ile Ala LysLys Val Phe Ser Leu Ile Gly Glu Gly Asp Tyr Asp 100 105 110 Lys Leu ArgAla Glu Phe Asp Lys Lys Ser Arg Tyr Arg Lys Leu Phe 115 120 125 Leu TrpLeu Asp Arg Ala Asp His Asp Arg Ile Leu Ser Trp Trp Arg 130 135 140 GlyTyr Ala Ala Lys Ser Lys Ile Pro Ser Asn Ala Leu Phe Phe Met 145 150 155160 Thr Asp Tyr Gln Arg Ser Tyr Pro Phe Gly Lys Leu Leu Gly Gln Val 165170 175 Leu His Thr Leu Arg Glu Val Lys Asp Glu Lys Thr Gly Lys Ala Phe180 185 190 Pro Thr Gly Gly Leu Glu Ala Tyr Phe Asn His Val Leu Glu GlyGlu 195 200 205 Pro Gly Glu Arg Lys Phe Leu Arg Ser Pro Leu Asn Arg LeuAsp Leu 210 215 220 Asp Lys Val Thr Lys Ile Pro Arg Asp Gly Ser Asp IleTyr Leu Thr 225 230 235 240 Val Asn Pro Cys Ile Gln Thr Ile Ala Glu GluGlu Leu Glu Lys Gly 245 250 255 Val Lys Glu Ala Lys Ala Lys Gly Gly ArgLeu Ile Leu Met Asn Ala 260 265 270 Tyr Thr Gly Glu Ile Leu Ala Leu AlaGln Tyr Pro Phe Phe Asn Pro 275 280 285 Ser Glu Tyr Lys Glu Phe Phe AsnAsp Lys Glu Lys Ile Glu His Thr 290 295 300 Lys Val Thr Ser Val Ser AspVal Phe Glu Pro Gly Ser Ile Met Lys 305 310 315 320 Pro Leu Thr Leu AlaIle Ala Leu Leu Ala Asn Glu Glu Met Val Lys 325 330 335 Arg Ser Gly LysPro Leu Phe Asp Pro Asn Glu Pro Ile Asp Val Thr 340 345 350 Arg Arg IlePhe Pro Gly Arg Lys Gln Phe Pro Leu Lys Asp Ile Ser 355 360 365 Ser AsnArg Arg Leu Asn Met Tyr Met Ala Ile Gln Lys Ser Ser Asn 370 375 380 ValTyr Val Ala Gln Leu Ala Asp Leu Ile Val Gln His Leu Gly Asn 385 390 395400 His Trp Tyr Glu Asp Lys Leu Leu Leu Leu Gly Phe Gly Lys Lys Thr 405410 415 Gly Ile Glu Leu Pro Gly Glu Ala Ser Gly Leu Val Pro Ser Pro Lys420 425 430 Arg Phe His Ile Asn Gly Val Pro Glu Trp Ser Leu Ser Thr ProTyr 435 440 445 Ser Leu Ala Met Gly Tyr Asn Ile Leu Ala Thr Gly Val GlnMet Val 450 455 460 Lys Ala Tyr Ala Ile Leu Ala Asn Gly Gly Tyr Asp ValArg Pro Thr 465 470 475 480 Leu Ile Lys Lys Ile Val Thr Thr Ser Gly LysGlu Tyr Val Leu His 485 490 495 Pro Gln Val Arg Gly Glu Arg Ile Leu SerGln Asp Ile Val Asp Glu 500 505 510 Val Leu Lys Ala Thr Arg Phe Thr ThrTyr Pro Gly Gly Thr Gly Phe 515 520 525 Arg Ala Ala Pro Lys Lys His SerSer Ala Gly Lys Thr Gly Thr Thr 530 535 540 Glu Lys Leu Val His Gly LysTyr Asp Lys His Arg His Ile Ser Ser 545 550 555 560 Phe Ile Gly Ile ThrPro Ile Tyr Pro Ser Ala Gly Gly Ser Val Pro 565 570 575 Leu Val Met LeuVal Ser Ile Asp Asp Pro Asp His Cys Val Arg Glu 580 585 590 Asp Gly ThrLys Asn Tyr Met Gly Gly Arg Cys Ala Ala Pro Val Phe 595 600 605 Gly ArgVal Ala Asp Arg Val Leu Ser Tyr Leu Gly Val Pro Glu Asp 610 615 620 LysGlu Lys Tyr Ser Tyr Gln Ser Glu Val Ala Ala Met Lys Ala Leu 625 630 635640 Tyr Glu Glu Trp Asn Arg Ser Gly Lys 645 28 960 DNA Chlamydiapsittaci 28 atgttcaata agctcattga aacagcacag aaacgggtgg aagcaagaaactatactatt 60 cgaaagcata ctcttgagta tgacgatgtt atgaataggc aaaggcagacgatctatgct 120 tttcgtaatg acgttatccg ctctgaagat atctttggtt tagctaaggaagcaatatct 180 catgttgcat taatgatcgc ttcgttgata gtgagccgtg atcatcctacagggaattct 240 cttcctaggc tggaagaatg gatgaactat tctttcccac tgcaattgaatattgaagaa 300 ttgaaaagat tgaagtctat agatgccatt gccgaacggg ttgctgatgatctcatagaa 360 gttttccaga ataagtttgc ttctatggtg caggaaatta ccgaagcagccggagaaaaa 420 gtcgatgcta atggtgtctg taaagatgtt attcgctcgg tcatgattatgcatatcgat 480 gagcagtgga aaattcatct tgtagatatg gatttattac gtagtgaagtaggtttacgt 540 actgtcggtc agaaagaccc tcttatcgaa tttaaacatg agtcgttcttactattcgaa 600 agtcttattc gcgatattcg tattgctatt gtaaagcatt tgttccgtttagagttgacg 660 atgactagag aacagcggcc tcaaaatgtc gtgcctgttg ttgccacatctttccaaaat 720 aatgaaaatt tcggtccttt ggaactcaca gttatcagtg attctgacgatgaataaaaa 780 gagctttagg gctgggctag cttccagcct tttcccttac gttattgatttatagtttta 840 aataaatacg gaccactcag accaggattg tgtgtcgtgg tggcgtatccaaaatgttct 900 gtgattatcc tcaatcagaa attgtacatg atgatcgcga ttgcgtgttgtcatgcaaat 960 29 258 PRT Chlamydia psittaci 29 Met Phe Asn Lys Leu IleGlu Thr Ala Gln Lys Arg Val Glu Ala Arg 1 5 10 15 Asn Tyr Thr Ile ArgLys His Thr Leu Glu Tyr Asp Asp Val Met Asn 20 25 30 Arg Gln Arg Gln ThrIle Tyr Ala Phe Arg Asn Asp Val Ile Arg Ser 35 40 45 Glu Asp Ile Phe GlyLeu Ala Lys Glu Ala Ile Ser His Val Ala Leu 50 55 60 Met Ile Ala Ser LeuIle Val Ser Arg Asp His Pro Thr Gly Asn Ser 65 70 75 80 Leu Pro Arg LeuGlu Glu Trp Met Asn Tyr Ser Phe Pro Leu Gln Leu 85 90 95 Asn Ile Glu GluLeu Lys Arg Leu Lys Ser Ile Asp Ala Ile Ala Glu 100 105 110 Arg Val AlaAsp Asp Leu Ile Glu Val Phe Gln Asn Lys Phe Ala Ser 115 120 125 Met ValGln Glu Ile Thr Glu Ala Ala Gly Glu Lys Val Asp Ala Asn 130 135 140 GlyVal Cys Lys Asp Val Ile Arg Ser Val Met Ile Met His Ile Asp 145 150 155160 Glu Gln Trp Lys Ile His Leu Val Asp Met Asp Leu Leu Arg Ser Glu 165170 175 Val Gly Leu Arg Thr Val Gly Gln Lys Asp Pro Leu Ile Glu Phe Lys180 185 190 His Glu Ser Phe Leu Leu Phe Glu Ser Leu Ile Arg Asp Ile ArgIle 195 200 205 Ala Ile Val Lys His Leu Phe Arg Leu Glu Leu Thr Met ThrArg Glu 210 215 220 Gln Arg Pro Gln Asn Val Val Pro Val Val Ala Thr SerPhe Gln Asn 225 230 235 240 Asn Glu Asn Phe Gly Pro Leu Glu Leu Thr ValIle Ser Asp Ser Asp 245 250 255 Asp Glu 30 697 DNA Chlamydia psittaci 30gggtttgatt atctcagaga taattctatt gcaacttctg tggatgagca ggtgggacgt 60gggttttatt ttgctattat cgatgaagtc gactcgattt taattgatga agccagaact 120cctttaatta tttctggtcc tggggaaaaa cataatcctg tgtatttcga actcaaagat 180aaagtggctg acctcgttca gttacaaagg gagttatgta accagttagc tcttgaagct 240agacggggac tagaattgtt cttggatatg gatattcttc ctaaggataa aaaagttatc 300gaagctatct ccgaattttg ccgtagctta tggttagtta gtaagggaat gcctttaaat 360cgtgttttgc gtagagtgcg cgaacaccca gatttgcgag ccatgataga taaatgggat 420acttattatc atgctgagca aaataaagaa gagagtatag agaagctatc tcagctgtat 480atcattgttg atgaacataa taacgatttt gaattgacag atcgtggcat gcaacaatgg 540gtggataagg ctggaggttc tgctgaagat tttgtcatga tggacatggg gcatgaatat 600gctcttatag atggtgacga taccttatca ccgacagaga aaatcaatag aaaaatagct 660atttccgaag aagatacgag gagaaaagct cgagctc 697 31 232 PRT Chlamydiapsittaci 31 Gly Phe Asp Tyr Leu Arg Asp Asn Ser Ile Ala Thr Ser Val AspGlu 1 5 10 15 Gln Val Gly Arg Gly Phe Tyr Phe Ala Ile Ile Asp Glu ValAsp Ser 20 25 30 Ile Leu Ile Asp Glu Ala Arg Thr Pro Leu Ile Ile Ser GlyPro Gly 35 40 45 Glu Lys His Asn Pro Val Tyr Phe Glu Leu Lys Asp Lys ValAla Asp 50 55 60 Leu Val Gln Leu Gln Arg Glu Leu Cys Asn Gln Leu Ala LeuGlu Ala 65 70 75 80 Arg Arg Gly Leu Glu Leu Phe Leu Asp Met Asp Ile LeuPro Lys Asp 85 90 95 Lys Lys Val Ile Glu Ala Ile Ser Glu Phe Cys Arg SerLeu Trp Leu 100 105 110 Val Ser Lys Gly Met Pro Leu Asn Arg Val Leu ArgArg Val Arg Glu 115 120 125 His Pro Asp Leu Arg Ala Met Ile Asp Lys TrpAsp Thr Tyr Tyr His 130 135 140 Ala Glu Gln Asn Lys Glu Glu Ser Ile GluLys Leu Ser Gln Leu Tyr 145 150 155 160 Ile Ile Val Asp Glu His Asn AsnAsp Phe Glu Leu Thr Asp Arg Gly 165 170 175 Met Gln Gln Trp Val Asp LysAla Gly Gly Ser Ala Glu Asp Phe Val 180 185 190 Met Met Asp Met Gly HisGlu Tyr Ala Leu Ile Asp Gly Asp Asp Thr 195 200 205 Leu Ser Pro Thr GluLys Ile Asn Arg Lys Ile Ala Ile Ser Glu Glu 210 215 220 Asp Thr Arg ArgLys Ala Arg Ala 225 230 32 2910 DNA Chlamydia psittaci 32 atgttagattttcttaaacg tttctttgga tcttctcaag agcgcacctt aaaaaaattt 60 caaaaacttgtggataaggt caacctctat gatgagatgc tagctccttt gtctgatgag 120 gagttacgtaataaaacagc agagttaaaa aagcgttatc aggacggcga atccttagat 180 gatatgcttcccgaggctta tgccgtagtg aaaaatgtat gcaggcgttt aacaggaact 240 cctgtagaagtgtcgggtta tcatcaaaat tgggacatgg ttccctatga tgtgcaggtt 300 ctcggtgctatagctatgca taagggcttt ataaccgaga tgcagacagg agaggggaaa 360 actctcaccgctgttatgcc tctatattta aatgcattga caggcaagcc tgtgcattta 420 gtcacagtgaatgattatct cgctcaaagg gattgtgagt gggtcggctc tatattgcgt 480 tggttaggtttaactaccgg agtattgata tcaggatcgc ctttagaaaa aagaaaagac 540 atttatcgttgtgacgttgt ctacggtaca gcatcagagt tcgggtttga ttatctcaga 600 gataattctattgcaacttc tgtggatgag caggtgggac gtgggtttta ttttgctatt 660 atcgatgaagtcgactcgat tttaattgat gaagccagaa ctcctttaat tatttctggt 720 cctggggaaaaacataatcc tgtgtatttc gaactcaaag ataaagtggc tgacctcgtt 780 cagttacaaagggagttatg taaccagtta gctcttgaag ctagacgggg actagaattg 840 ttcttggatatggatattct tcctaaggat aaaaaagtta tcgaagctat ctccgaattt 900 tgccgtagcttatggttagt tagtaaggga atgcctttaa atcgtgtttt gcgtagagtg 960 cgcgaacacccagatttgcg agccatgata gataaatggg atacttatta tcatgctgag 1020 caaaataaagaagagagtat agagaagcta tctcagctgt atatcattgt tgatgaacat 1080 aataacgattttgaattgac agatcgtggc atgcaacaat gggtggataa ggctggaggt 1140 tctgctgaagattttgtcat gatggacatg gggcatgaat atgctcttat agatggtgac 1200 gataccttatcaccgacaga gaaaatcaat agaaaaatag ctatttccga agaagatacg 1260 aggagaaaagctcgagctca tggcttgcgc caactattaa gagcgcatct tcttatggaa 1320 cgcgatgtggattatattgt tcgtaatgat caaattgtca tcattgacga acatacgggc 1380 cgcccgcaaccaggtcgtcg tttttccgaa ggactgcatc aagccataga agcaaaagaa 1440 catgtcactatccgtaagga atcacaaacg tttgctacag ttaccttaca gaatttcttc 1500 cgtctgtatgaaaaactcgc aggtatgacg ggaacagcaa ttacggaatc taaagagttt 1560 aaagagatttataatcttta tgtattgcag gtgcccacgt ttaaagaatg tttgcgtgta 1620 gatcacaatgacgaatttta tatgacagag cgtgaaaagt accacgcgat tgttaaggaa 1680 attgcccgtatacatgccgt agggaacccg attctcatag gaacggagtc tgtagaggtt 1740 tctgagaaactttctcgtat tttgagacaa aatcgcatag aacatacagt gttaaatgcg 1800 aaaaatcatgctcaagaagc agagatcatt gcagcagcag gaaagctggg agctgtgact 1860 gtagctaccaatatggctgg ccgtggtaca gatattaagc tggatgaaga agctgtagtt 1920 gttggaggtctccatgttat tggtacgagt cggcaccaat cacgccgtat agataggcag 1980 ttgcgcgggcgttgcgcacg tttaggagat cctggttcgg cgaaattttt cctatctttt 2040 gaagatcgcctgatgcgctt atttgcatcg cccaagttaa atgccttgat tcgtcatttc 2100 cgtcctcctgaaggagaggc tatgtcggat cctatgttca ataagctcat tgaaacagca 2160 cagaaacgggtggaagcaag aaactatact attcgaaagc atactcttga gtatgacgat 2220 gttatgaataggcaaaggca gacgatctat gcttttcgta atgacgttat ccgctctgaa 2280 gatatctttggtttagctaa ggaagcaata tctcatgttg cattaatgat cgcttcgttg 2340 atagtgagccgtgatcatcc tacagggaat tctcttccta ggctggaaga atggatgaac 2400 tattctttcccactgcaatt gaatattgaa gaattgaaaa gattgaagtc tatagatgcc 2460 attgccgaacgggttgctga tgatctcata gaagttttcc agaataagtt tgcttctatg 2520 gtgcaggaaattaccgaagc agccggagaa aaagtcgatg ctaatggtgt ctgtaaagat 2580 gttattcgctcggtcatgat tatgcatatc gatgagcagt ggaaaattca tcttgtagat 2640 atggatttattacgtagtga agtaggttta cgtactgtcg gtcagaaaga ccctcttatc 2700 gaatttaaacatgagtcgtt cttactattc gaaagtctta ttcgcgatat tcgtattgct 2760 attgtaaagcatttgttccg tttagagttg acgatgacta gagaacagcg gcctcaaaat 2820 gtcgtgcctgttgttgccac atctttccaa aataatgaaa atttcggtcc tttggaactc 2880 acagttatcagtgattctga cgatgaataa 2910 33 969 PRT Chlamydia psittaci 33 Met Leu AspPhe Leu Lys Arg Phe Phe Gly Ser Ser Gln Glu Arg Thr 1 5 10 15 Leu LysLys Phe Gln Lys Leu Val Asp Lys Val Asn Leu Tyr Asp Glu 20 25 30 Met LeuAla Pro Leu Ser Asp Glu Glu Leu Arg Asn Lys Thr Ala Glu 35 40 45 Leu LysLys Arg Tyr Gln Asp Gly Glu Ser Leu Asp Asp Met Leu Pro 50 55 60 Glu AlaTyr Ala Val Val Lys Asn Val Cys Arg Arg Leu Thr Gly Thr 65 70 75 80 ProVal Glu Val Ser Gly Tyr His Gln Asn Trp Asp Met Val Pro Tyr 85 90 95 AspVal Gln Val Leu Gly Ala Ile Ala Met His Lys Gly Phe Ile Thr 100 105 110Glu Met Gln Thr Gly Glu Gly Lys Thr Leu Thr Ala Val Met Pro Leu 115 120125 Tyr Leu Asn Ala Leu Thr Gly Lys Pro Val His Leu Val Thr Val Asn 130135 140 Asp Tyr Leu Ala Gln Arg Asp Cys Glu Trp Val Gly Ser Ile Leu Arg145 150 155 160 Trp Leu Gly Leu Thr Thr Gly Val Leu Ile Ser Gly Ser ProLeu Glu 165 170 175 Lys Arg Lys Asp Ile Tyr Arg Cys Asp Val Val Tyr GlyThr Ala Ser 180 185 190 Glu Phe Gly Phe Asp Tyr Leu Arg Asp Asn Ser IleAla Thr Ser Val 195 200 205 Asp Glu Gln Val Gly Arg Gly Phe Tyr Phe AlaIle Ile Asp Glu Val 210 215 220 Asp Ser Ile Leu Ile Asp Glu Ala Arg ThrPro Leu Ile Ile Ser Gly 225 230 235 240 Pro Gly Glu Lys His Asn Pro ValTyr Phe Glu Leu Lys Asp Lys Val 245 250 255 Ala Asp Leu Val Gln Leu GlnArg Glu Leu Cys Asn Gln Leu Ala Leu 260 265 270 Glu Ala Arg Arg Gly LeuGlu Leu Phe Leu Asp Met Asp Ile Leu Pro 275 280 285 Lys Asp Lys Lys ValIle Glu Ala Ile Ser Glu Phe Cys Arg Ser Leu 290 295 300 Trp Leu Val SerLys Gly Met Pro Leu Asn Arg Val Leu Arg Arg Val 305 310 315 320 Arg GluHis Pro Asp Leu Arg Ala Met Ile Asp Lys Trp Asp Thr Tyr 325 330 335 TyrHis Ala Glu Gln Asn Lys Glu Glu Ser Ile Glu Lys Leu Ser Gln 340 345 350Leu Tyr Ile Ile Val Asp Glu His Asn Asn Asp Phe Glu Leu Thr Asp 355 360365 Arg Gly Met Gln Gln Trp Val Asp Lys Ala Gly Gly Ser Ala Glu Asp 370375 380 Phe Val Met Met Asp Met Gly His Glu Tyr Ala Leu Ile Asp Gly Asp385 390 395 400 Asp Thr Leu Ser Pro Thr Glu Lys Ile Asn Arg Lys Ile AlaIle Ser 405 410 415 Glu Glu Asp Thr Arg Arg Lys Ala Arg Ala His Gly LeuArg Gln Leu 420 425 430 Leu Arg Ala His Leu Leu Met Glu Arg Asp Val AspTyr Ile Val Arg 435 440 445 Asn Asp Gln Ile Val Ile Ile Asp Glu His ThrGly Arg Pro Gln Pro 450 455 460 Gly Arg Arg Phe Ser Glu Gly Leu His GlnAla Ile Glu Ala Lys Glu 465 470 475 480 His Val Thr Ile Arg Lys Glu SerGln Thr Phe Ala Thr Val Thr Leu 485 490 495 Gln Asn Phe Phe Arg Leu TyrGlu Lys Leu Ala Gly Met Thr Gly Thr 500 505 510 Ala Ile Thr Glu Ser LysGlu Phe Lys Glu Ile Tyr Asn Leu Tyr Val 515 520 525 Leu Gln Val Pro ThrPhe Lys Glu Cys Leu Arg Val Asp His Asn Asp 530 535 540 Glu Phe Tyr MetThr Glu Arg Glu Lys Tyr His Ala Ile Val Lys Glu 545 550 555 560 Ile AlaArg Ile His Ala Val Gly Asn Pro Ile Leu Ile Gly Thr Glu 565 570 575 SerVal Glu Val Ser Glu Lys Leu Ser Arg Ile Leu Arg Gln Asn Arg 580 585 590Ile Glu His Thr Val Leu Asn Ala Lys Asn His Ala Gln Glu Ala Glu 595 600605 Ile Ile Ala Ala Ala Gly Lys Leu Gly Ala Val Thr Val Ala Thr Asn 610615 620 Met Ala Gly Arg Gly Thr Asp Ile Lys Leu Asp Glu Glu Ala Val Val625 630 635 640 Val Gly Gly Leu His Val Ile Gly Thr Ser Arg His Gln SerArg Arg 645 650 655 Ile Asp Arg Gln Leu Arg Gly Arg Cys Ala Arg Leu GlyAsp Pro Gly 660 665 670 Ser Ala Lys Phe Phe Leu Ser Phe Glu Asp Arg LeuMet Arg Leu Phe 675 680 685 Ala Ser Pro Lys Leu Asn Ala Leu Ile Arg HisPhe Arg Pro Pro Glu 690 695 700 Gly Glu Ala Met Ser Asp Pro Met Phe AsnLys Leu Ile Glu Thr Ala 705 710 715 720 Gln Lys Arg Val Glu Ala Arg AsnTyr Thr Ile Arg Lys His Thr Leu 725 730 735 Glu Tyr Asp Asp Val Met AsnArg Gln Arg Gln Thr Ile Tyr Ala Phe 740 745 750 Arg Asn Asp Val Ile ArgSer Glu Asp Ile Phe Gly Leu Ala Lys Glu 755 760 765 Ala Ile Ser His ValAla Leu Met Ile Ala Ser Leu Ile Val Ser Arg 770 775 780 Asp His Pro ThrGly Asn Ser Leu Pro Arg Leu Glu Glu Trp Met Asn 785 790 795 800 Tyr SerPhe Pro Leu Gln Leu Asn Ile Glu Glu Leu Lys Arg Leu Lys 805 810 815 SerIle Asp Ala Ile Ala Glu Arg Val Ala Asp Asp Leu Ile Glu Val 820 825 830Phe Gln Asn Lys Phe Ala Ser Met Val Gln Glu Ile Thr Glu Ala Ala 835 840845 Gly Glu Lys Val Asp Ala Asn Gly Val Cys Lys Asp Val Ile Arg Ser 850855 860 Val Met Ile Met His Ile Asp Glu Gln Trp Lys Ile His Leu Val Asp865 870 875 880 Met Asp Leu Leu Arg Ser Glu Val Gly Leu Arg Thr Val GlyGln Lys 885 890 895 Asp Pro Leu Ile Glu Phe Lys His Glu Ser Phe Leu LeuPhe Glu Ser 900 905 910 Leu Ile Arg Asp Ile Arg Ile Ala Ile Val Lys HisLeu Phe Arg Leu 915 920 925 Glu Leu Thr Met Thr Arg Glu Gln Arg Pro GlnAsn Val Val Pro Val 930 935 940 Val Ala Thr Ser Phe Gln Asn Asn Glu AsnPhe Gly Pro Leu Glu Leu 945 950 955 960 Thr Val Ile Ser Asp Ser Asp AspGlu 965 34 577 DNA Chlamydia psittaci 34 gttgatgctg cagttattccagggaacttc gccattgcag ggggaatctg tccgtataaa 60 aacagtctat acctagaagatgtccgtact tcccaataca ccaatgtcgt tgtcatacgt 120 gctgaagata tggaagactcgagaatgcat aaactaaaac agctattgca aagcagttct 180 gtgcaggatt tctttaatacgaaatataag gggatctttt tatcgcagta acacatctgg 240 atggcttagg gaagagttgagccaccccgt tctccgtagg tttaaggcat attgggaaac 300 gattttcttg aattttttgaaaaactttga ctgtttttct tttgattatt cgaagcagat 360 gtatgtcgag tatggcggttttagggccca gaggtccttt cagttctcct tttacatgtt 420 ctctataccc aacccacctaaaaatgcact tgctaggttc cattcctata gttggcatat 480 acattggagc gaagcggatagccgccgttg ctcaatatca tagaatgtgt agagcgaata 540 caggagtgtc tcaggtgattattcaggatt caggatt 577 35 76 PRT Chlamydia psittaci 35 Val Asp Ala AlaVal Ile Pro Gly Asn Phe Ala Ile Ala Gly Gly Ile 1 5 10 15 Cys Pro TyrLys Asn Ser Leu Tyr Leu Glu Asp Val Arg Thr Ser Gln 20 25 30 Tyr Thr AsnVal Val Val Ile Arg Ala Glu Asp Met Glu Asp Ser Arg 35 40 45 Met His LysLeu Lys Gln Leu Leu Gln Ser Ser Ser Val Gln Asp Phe 50 55 60 Phe Asn ThrLys Tyr Lys Gly Ile Phe Leu Ser Gln 65 70 75 36 804 DNA Chlamydiapsittaci 36 atgaaaaaaa tcacaatact ctcgttactt gctttagcca tctctttaacaggttgttgc 60 aagaattcag aaggagtctt gcggattgcg gcgagtccca cgccacatgcagagcttctt 120 tatagtttag aaaaggaggc tcaatccctt ggattgcaat tgaaaatacttcccatagat 180 gattaccgtg tacctaaccg tttgctttta gataagcaaa tagaggcaaattatttccaa 240 catgaagatt tcttaaaaga tgagtgtgct cggtaccaat gcgaaggaaaacttgcgatt 300 ttggctaagg tacatttaga acctatgggt ttatattcta ataaaacccagtctctcgaa 360 gagcttaaag tcaaggaaca gctacgtata gcggttccta tagatagaacaaacgaacaa 420 cgtgcgctag acttattgcg agactgcaat ttgattagtt acaaagaagcttctcatcta 480 gatatcaccg caaaagatgt ctttggttgt ggagggaaaa aggtaacgattatagagatg 540 gcagcacctt tattagtatc ttctttacca gacgttgatg ctgcagttattccagggaac 600 ttcgccattg cagggggaat ctgtccgtat aaaaacagtc tatacctagaagatgtccgt 660 acttcccaat acaccaatgt cgttgtcata cgtgctgaag atatggaagactcgagaatg 720 cataaactaa aacagctatt gcaaagcagt tctgtgcagg atttctttaatacgaaatat 780 aaggggatct ttttatcgca gtaa 804 37 267 PRT Chlamydiapsittaci 37 Met Lys Lys Ile Thr Ile Leu Ser Leu Leu Ala Leu Ala Ile SerLeu 1 5 10 15 Thr Gly Cys Cys Lys Asn Ser Glu Gly Val Leu Arg Ile AlaAla Ser 20 25 30 Pro Thr Pro His Ala Glu Leu Leu Tyr Ser Leu Glu Lys GluAla Gln 35 40 45 Ser Leu Gly Leu Gln Leu Lys Ile Leu Pro Ile Asp Asp TyrArg Val 50 55 60 Pro Asn Arg Leu Leu Leu Asp Lys Gln Ile Glu Ala Asn TyrPhe Gln 65 70 75 80 His Glu Asp Phe Leu Lys Asp Glu Cys Ala Arg Tyr GlnCys Glu Gly 85 90 95 Lys Leu Ala Ile Leu Ala Lys Val His Leu Glu Pro MetGly Leu Tyr 100 105 110 Ser Asn Lys Thr Gln Ser Leu Glu Glu Leu Lys ValLys Glu Gln Leu 115 120 125 Arg Ile Ala Val Pro Ile Asp Arg Thr Asn GluGln Arg Ala Leu Asp 130 135 140 Leu Leu Arg Asp Cys Asn Leu Ile Ser TyrLys Glu Ala Ser His Leu 145 150 155 160 Asp Ile Thr Ala Lys Asp Val PheGly Cys Gly Gly Lys Lys Val Thr 165 170 175 Ile Ile Glu Met Ala Ala ProLeu Leu Val Ser Ser Leu Pro Asp Val 180 185 190 Asp Ala Ala Val Ile ProGly Asn Phe Ala Ile Ala Gly Gly Ile Cys 195 200 205 Pro Tyr Lys Asn SerLeu Tyr Leu Glu Asp Val Arg Thr Ser Gln Tyr 210 215 220 Thr Asn Val ValVal Ile Arg Ala Glu Asp Met Glu Asp Ser Arg Met 225 230 235 240 His LysLeu Lys Gln Leu Leu Gln Ser Ser Ser Val Gln Asp Phe Phe 245 250 255 AsnThr Lys Tyr Lys Gly Ile Phe Leu Ser Gln 260 265 38 402 DNA Chlamydiapsittaci 38 catgtatttt acgcaaaaaa taaacggtat aactcctgct tacaagccgcgctataccac 60 aataatatcc cgacaaccgt gtacacaaac cttattgata tcgtgaagaaaaattcttca 120 ctaatcacga agtacttttc catcaaacaa cgatgcttaa atctaaaagatttccatttt 180 tatgatgttt atgctcccct aagtcagtcc aaagagaaaa aatatacgttccaagaagct 240 gtggatctta tctatactag cctttctcct ctaggaacgg aatacattgataccttaaaa 300 caggggttaa caactcaagg ctgggtagat aaatacgaaa atcttaataaacgctccgga 360 gcctattctt cgggatgtta cgatagccac ccttatgtcc tc 402 39 134PRT Chlamydia psittaci 39 His Val Phe Tyr Ala Lys Asn Lys Arg Tyr AsnSer Cys Leu Gln Ala 1 5 10 15 Ala Leu Tyr His Asn Asn Ile Pro Thr ThrVal Tyr Thr Asn Leu Ile 20 25 30 Asp Ile Val Lys Lys Asn Ser Ser Leu IleThr Lys Tyr Phe Ser Ile 35 40 45 Lys Gln Arg Cys Leu Asn Leu Lys Asp PheHis Phe Tyr Asp Val Tyr 50 55 60 Ala Pro Leu Ser Gln Ser Lys Glu Lys LysTyr Thr Phe Gln Glu Ala 65 70 75 80 Val Asp Leu Ile Tyr Thr Ser Leu SerPro Leu Gly Thr Glu Tyr Ile 85 90 95 Asp Thr Leu Lys Gln Gly Leu Thr ThrGln Gly Trp Val Asp Lys Tyr 100 105 110 Glu Asn Leu Asn Lys Arg Ser GlyAla Tyr Ser Ser Gly Cys Tyr Asp 115 120 125 Ser His Pro Tyr Val Leu 13040 1827 DNA Chlamydia psittaci 40 atgagcgtag aattcaacaa gcaacaagtccgtccaagaa gtgaaatttc ccctcaagat 60 tgttgggata tcaccccctt atatctaaatagaaaagcat ggaaagcaga tcttgattct 120 ttcggattaa aaacagacgg ctcacctacgtggcccgctc ttcaagcaac gcaataccaa 180 ctggacaact cagaatctct actatccttattaactactc tcttctctat tgagagaaaa 240 ttaaacaaac tctacgttta cgctcatctgactcatgatc aggatattac aaatcaagaa 300 ggcatcgcag atcttaaatc tatcacgcatctacatacct tatttgccga agaaacctct 360 tgggtacaac ccgctttaac cagcctatcggaatctctca ttgctcagca cctatcagct 420 ccctgtttag ctccttatag attctatttagagaaaatct ttagactatc tatacacaca 480 ggcactcctg gagaagaaaa aattctcgcttccgccttta ctcctcttga agtagccagt 540 aaggcatttt cttctttaag tgactctgaaattccctttg ggcaagctac agactcagaa 600 ggaaactctc acccgctttc tcatgcactggcttcattgt atatgcaatc cacagatcgg 660 gaattacgaa aaacatccta cctagcacaatgtgaaagat atcatagtta ccgacatacc 720 tttgctaact tactcaatgg gaaaatccaagcccatgtat tttacgcaaa aaataaacgg 780 tataactcct gcttacaagc cgcgctataccacaataata tcccgacaac cgtgtacaca 840 aaccttattg atatcgtgaa gaaaaattcttcactaatca cgaagtactt ttccatcaaa 900 caacgatgct taaatctaaa agatttccatttttatgatg tttatgctcc cctaagtcag 960 tccaaagaga aaaaatatac gttccaagaagctgtggatc ttatctatac tagcctttct 1020 cctctaggaa cggaatacat tgataccttaaaacaggggt taacaactca aggctgggta 1080 gataaatacg aaaatcttaa taaacgctccggagcctatt cttcgggatg ttacgatagc 1140 cacccttatg tcctcctaaa ctatacaggcaccctgtatg atgtatccgt cattgcccac 1200 gaaggcggac acagtatgca ctcgtattttagtaggaagc atcaaccttt ccatgacgct 1260 caatatccta ttttccttgc tgaaattgcttctaccttaa atgaaatgct tcttatggat 1320 tccatgctga aggagagcga ctcaaaagaagagaaaatca ccattctgac acgatgtttg 1380 gataccatct tctctacact attccgtcaggtattattcg cctcttttga atacgatatt 1440 catcacgcag cagaacatgg ggttcctctaactgaagaat acctatcctc aacttacaag 1500 aatttacaaa atgagtttta cggagaaattatcacatttg atgtcctgtc gaacatagaa 1560 tgggcaagaa ttcctcattt ctattacaatttctacgtat accaatatgc aacgggcatt 1620 atagccgccc tgtgcttttt agaaaaaattcttaacaacg aagataacgc tcttaactcc 1680 tatctcaact ttttaaaaag tggtggatcagatttcccct tagaaatctt aaaaaaatca 1740 ggattagata tgggcacagt tgagccaatccaaaaagctt tttgctttat cgagaaaaaa 1800 atccaggagc tatcatcttt aatttga 182741 608 PRT Chlamydia psittaci 41 Met Ser Val Glu Phe Asn Lys Gln Gln ValArg Pro Arg Ser Glu Ile 1 5 10 15 Ser Pro Gln Asp Cys Trp Asp Ile ThrPro Leu Tyr Leu Asn Arg Lys 20 25 30 Ala Trp Lys Ala Asp Leu Asp Ser PheGly Leu Lys Thr Asp Gly Ser 35 40 45 Pro Thr Trp Pro Ala Leu Gln Ala ThrGln Tyr Gln Leu Asp Asn Ser 50 55 60 Glu Ser Leu Leu Ser Leu Leu Thr ThrLeu Phe Ser Ile Glu Arg Lys 65 70 75 80 Leu Asn Lys Leu Tyr Val Tyr AlaHis Leu Thr His Asp Gln Asp Ile 85 90 95 Thr Asn Gln Glu Gly Ile Ala AspLeu Lys Ser Ile Thr His Leu His 100 105 110 Thr Leu Phe Ala Glu Glu ThrSer Trp Val Gln Pro Ala Leu Thr Ser 115 120 125 Leu Ser Glu Ser Leu IleAla Gln His Leu Ser Ala Pro Cys Leu Ala 130 135 140 Pro Tyr Arg Phe TyrLeu Glu Lys Ile Phe Arg Leu Ser Ile His Thr 145 150 155 160 Gly Thr ProGly Glu Glu Lys Ile Leu Ala Ser Ala Phe Thr Pro Leu 165 170 175 Glu ValAla Ser Lys Ala Phe Ser Ser Leu Ser Asp Ser Glu Ile Pro 180 185 190 PheGly Gln Ala Thr Asp Ser Glu Gly Asn Ser His Pro Leu Ser His 195 200 205Ala Leu Ala Ser Leu Tyr Met Gln Ser Thr Asp Arg Glu Leu Arg Lys 210 215220 Thr Ser Tyr Leu Ala Gln Cys Glu Arg Tyr His Ser Tyr Arg His Thr 225230 235 240 Phe Ala Asn Leu Leu Asn Gly Lys Ile Gln Ala His Val Phe TyrAla 245 250 255 Lys Asn Lys Arg Tyr Asn Ser Cys Leu Gln Ala Ala Leu TyrHis Asn 260 265 270 Asn Ile Pro Thr Thr Val Tyr Thr Asn Leu Ile Asp IleVal Lys Lys 275 280 285 Asn Ser Ser Leu Ile Thr Lys Tyr Phe Ser Ile LysGln Arg Cys Leu 290 295 300 Asn Leu Lys Asp Phe His Phe Tyr Asp Val TyrAla Pro Leu Ser Gln 305 310 315 320 Ser Lys Glu Lys Lys Tyr Thr Phe GlnGlu Ala Val Asp Leu Ile Tyr 325 330 335 Thr Ser Leu Ser Pro Leu Gly ThrGlu Tyr Ile Asp Thr Leu Lys Gln 340 345 350 Gly Leu Thr Thr Gln Gly TrpVal Asp Lys Tyr Glu Asn Leu Asn Lys 355 360 365 Arg Ser Gly Ala Tyr SerSer Gly Cys Tyr Asp Ser His Pro Tyr Val 370 375 380 Leu Leu Asn Tyr ThrGly Thr Leu Tyr Asp Val Ser Val Ile Ala His 385 390 395 400 Glu Gly GlyHis Ser Met His Ser Tyr Phe Ser Arg Lys His Gln Pro 405 410 415 Phe HisAsp Ala Gln Tyr Pro Ile Phe Leu Ala Glu Ile Ala Ser Thr 420 425 430 LeuAsn Glu Met Leu Leu Met Asp Ser Met Leu Lys Glu Ser Asp Ser 435 440 445Lys Glu Glu Lys Ile Thr Ile Leu Thr Arg Cys Leu Asp Thr Ile Phe 450 455460 Ser Thr Leu Phe Arg Gln Val Leu Phe Ala Ser Phe Glu Tyr Asp Ile 465470 475 480 His His Ala Ala Glu His Gly Val Pro Leu Thr Glu Glu Tyr LeuSer 485 490 495 Ser Thr Tyr Lys Asn Leu Gln Asn Glu Phe Tyr Gly Glu IleIle Thr 500 505 510 Phe Asp Val Leu Ser Asn Ile Glu Trp Ala Arg Ile ProHis Phe Tyr 515 520 525 Tyr Asn Phe Tyr Val Tyr Gln Tyr Ala Thr Gly IleIle Ala Ala Leu 530 535 540 Cys Phe Leu Glu Lys Ile Leu Asn Asn Glu AspAsn Ala Leu Asn Ser 545 550 555 560 Tyr Leu Asn Phe Leu Lys Ser Gly GlySer Asp Phe Pro Leu Glu Ile 565 570 575 Leu Lys Lys Ser Gly Leu Asp MetGly Thr Val Glu Pro Ile Gln Lys 580 585 590 Ala Phe Cys Phe Ile Glu LysLys Ile Gln Glu Leu Ser Ser Leu Ile 595 600 605 42 1517 DNA Chlamydiapsittaci 42 gcgttagatt cggaagagct gaaagagcaa ataaacaatc taaaagagcgtttatgggat 60 gcacaatcca ctctacaaca agatcaaaat aaactatcgc aagaacattttgaagctgtc 120 agtgtgatca ttgatttaat caatggtgat ctgaatgata tagctgagcatacgcaacaa 180 aacttacaaa ccaaaaaaga agaagaacac gagtccgttg cccgtaagatggtcaattgg 240 gtgtcttctg gagaagaagt gttaaataga gcccttctct acttctcagataggaatgga 300 gaacgggaaa atttagcaga ctttttaaaa gtacagtatg ctgttcaaagagcaacgcaa 360 agagcagaac tttttgctag tatcgtagga actacggtaa gtagtataaagacgataatg 420 accacacaat taggttaaca tggacgaatt gacgacagat ttcgataccctcatgtcgca 480 attgaacgac gtacacttga ctaccgttgt cggtcgtata actgaagtcgtcggtatgtt 540 aattaaagct gtcgttccca atgtacgcgt tggggaggta tgcttagttaaacgttatgg 600 tatggagccg ctcgtgaccg aagtcgtcgg cttcacacaa aatttcgcttttttatcgcc 660 actaggagaa cttactggag tcagcccttc ttcagaggtt attcccacaggtctgccttt 720 gtatatccgt gcaggtaacg gtcttttagg tcgtgtattg aatggtctgggagaacctat 780 cgactccgag atcaaaggac ctttggttga tgttaacgaa acctaccctgtgtttcgcgc 840 tccaccagat ccattgcata gagaaaaatt aagaacaatt ttatccaccggcgtgcggtg 900 tatcgacggt atgctcacag tcgccagagg ccagcgtata ggcatttttgctggagctgg 960 ggtgggtaaa tcgtctctct tgggaatgat cgctagaaac gcggaagaagccgatgtcaa 1020 tgtgattgct ctcatcggag agcggggccg agaggttcgt gaatttatcgagggcgatct 1080 cggagaagaa ggaatgaaac gttcggtgat cgtcgtctct acttcagatcaatcctcaca 1140 gttgcgatta aatgctgctt acgtaggcac cgctatagca gagtattttcgtgatcaggg 1200 caaaaccgta gttttgatga tggattctgt cacccgattt gcccgagccctaagagaagt 1260 cggtctagct gccggagaac cgccagctcg aggaggatac acaccttctgtattctcaac 1320 tttgcctagg ttattagaac gttccggagc ttcggataaa ggaacaatcacagcctttta 1380 cacagtactt gttgccgggg atgatatgaa tgaaccggtc gctgatgaagttaaatcgat 1440 tcttgatggt cacgttgtct tgtctaacgc tttagctcag gcataccattatcctgctat 1500 tgatgtctta gcatcta 1517 43 145 PRT Chlamydia psittaci 43Ala Leu Asp Ser Glu Glu Leu Lys Glu Gln Ile Asn Asn Leu Lys Glu 1 5 1015 Arg Leu Trp Asp Ala Gln Ser Thr Leu Gln Gln Asp Gln Asn Lys Leu 20 2530 Ser Gln Glu His Phe Glu Ala Val Ser Val Ile Ile Asp Leu Ile Asn 35 4045 Gly Asp Leu Asn Asp Ile Ala Glu His Thr Gln Gln Asn Leu Gln Thr 50 5560 Lys Lys Glu Glu Glu His Glu Ser Val Ala Arg Lys Met Val Asn Trp 65 7075 80 Val Ser Ser Gly Glu Glu Val Leu Asn Arg Ala Leu Leu Tyr Phe Ser 8590 95 Asp Arg Asn Gly Glu Arg Glu Asn Leu Ala Asp Phe Leu Lys Val Gln100 105 110 Tyr Ala Val Gln Arg Ala Thr Gln Arg Ala Glu Leu Phe Ala SerIle 115 120 125 Val Gly Thr Thr Val Ser Ser Ile Lys Thr Ile Met Thr ThrGln Leu 130 135 140 Gly 145 44 669 DNA Chlamydia psittaci 44 atggtagatcctttgaagct tttcccaaag ctagactccg agaaagaaac agcttctata 60 cagaagcctttaggaactcc tttagccagt gagttacata aggaagttcc tgcattttct 120 ttagggacggcagcagactc cttgaataaa aatatagagg atgtcaagcc taaccctatg 180 gcgatgatgcaagacagaaa ctctaacatt atcgatcctg aactggaaga ggcgttagat 240 tcggaagagctgaaagagca aataaacaat ctaaaagagc gtttatggga tgcacaatcc 300 actctacaacaagatcaaaa taaactatcg caagaacatt ttgaagctgt cagtgtgatc 360 attgatttaatcaatggtga tctgaatgat atagctgagc atacgcaaca aaacttacaa 420 accaaaaaagaagaagaaca cgagtccgtt gcccgtaaga tggtcaattg ggtgtcttct 480 ggagaagaagtgttaaatag agcccttctc tacttctcag ataggaatgg agaacgggaa 540 aatttagcagactttttaaa agtacagtat gctgttcaaa gagcaacgca aagagcagaa 600 ctttttgctagtatcgtagg aactacggta agtagtataa agacgataat gaccacacaa 660 ttaggttaa 66945 222 PRT Chlamydia psittaci 45 Met Val Asp Pro Leu Lys Leu Phe Pro LysLeu Asp Ser Glu Lys Glu 1 5 10 15 Thr Ala Ser Ile Gln Lys Pro Leu GlyThr Pro Leu Ala Ser Glu Leu 20 25 30 His Lys Glu Val Pro Ala Phe Ser LeuGly Thr Ala Ala Asp Ser Leu 35 40 45 Asn Lys Asn Ile Glu Asp Val Lys ProAsn Pro Met Ala Met Met Gln 50 55 60 Asp Arg Asn Ser Asn Ile Ile Asp ProGlu Leu Glu Glu Ala Leu Asp 65 70 75 80 Ser Glu Glu Leu Lys Glu Gln IleAsn Asn Leu Lys Glu Arg Leu Trp 85 90 95 Asp Ala Gln Ser Thr Leu Gln GlnAsp Gln Asn Lys Leu Ser Gln Glu 100 105 110 His Phe Glu Ala Val Ser ValIle Ile Asp Leu Ile Asn Gly Asp Leu 115 120 125 Asn Asp Ile Ala Glu HisThr Gln Gln Asn Leu Gln Thr Lys Lys Glu 130 135 140 Glu Glu His Glu SerVal Ala Arg Lys Met Val Asn Trp Val Ser Ser 145 150 155 160 Gly Glu GluVal Leu Asn Arg Ala Leu Leu Tyr Phe Ser Asp Arg Asn 165 170 175 Gly GluArg Glu Asn Leu Ala Asp Phe Leu Lys Val Gln Tyr Ala Val 180 185 190 GlnArg Ala Thr Gln Arg Ala Glu Leu Phe Ala Ser Ile Val Gly Thr 195 200 205Thr Val Ser Ser Ile Lys Thr Ile Met Thr Thr Gln Leu Gly 210 215 220 461329 DNA Chlamydia psittaci 46 atggacgaat tgacgacaga tttcgataccctcatgtcgc aattgaacga cgtacacttg 60 actaccgttg tcggtcgtat aactgaagtcgtcggtatgt taattaaagc tgtcgttccc 120 aatgtacgcg ttggggaggt atgcttagttaaacgttatg gtatggagcc gctcgtgacc 180 gaagtcgtcg gcttcacaca aaatttcgcttttttatcgc cactaggaga acttactgga 240 gtcagccctt cttcagaggt tattcccacaggtctgcctt tgtatatccg tgcaggtaac 300 ggtcttttag gtcgtgtatt gaatggtctgggagaaccta tcgactccga gatcaaagga 360 cctttggttg atgttaacga aacctaccctgtgtttcgcg ctccaccaga tccattgcat 420 agagaaaaat taagaacaat tttatccaccggcgtgcggt gtatcgacgg tatgctcaca 480 gtcgccagag gccagcgtat aggcatttttgctggagctg gggtgggtaa atcgtctctc 540 ttgggaatga tcgctagaaa cgcggaagaagccgatgtca atgtgattgc tctcatcgga 600 gagcggggcc gagaggttcg tgaatttatcgagggcgatc tcggagaaga aggaatgaaa 660 cgttcggtga tcgtcgtctc tacttcagatcaatcctcac agttgcgatt aaatgctgct 720 tacgtaggca ccgctatagc agagtattttcgtgatcagg gcaaaaccgt agttttgatg 780 atggattctg tcacccgatt tgcccgagccctaagagaag tcggtctagc tgccggagaa 840 ccgccagctc gaggaggata cacaccttctgtattctcaa ctttgcctag gttattagaa 900 cgttccggag cttcggataa aggaacaatcacagcctttt acacagtact tgttgccggg 960 gatgatatga atgaaccggt cgctgatgaagttaaatcga ttcttgatgg tcacgttgtc 1020 ttgtctaacg ctttagctca ggcataccattatcctgcta ttgatgtctt agcatctatc 1080 agccgattgc tgacagcaat tgttcctgaggaacaacgac gcatcatagg aaaagcccga 1140 gaggtgctgg caaaatacaa agcaaacgaaatgcttatac gtattggaga atatcgccga 1200 gggtccgatc gtgaagtgga ttttgctatagatcacattg ataaattgaa cagattctta 1260 aagcaagata ttcatgaaaa aacaaattacgaggaagcct cgcaacagct tcgggctatt 1320 ttccgataa 1329 47 442 PRTChlamydia psittaci 47 Met Asp Glu Leu Thr Thr Asp Phe Asp Thr Leu MetSer Gln Leu Asn 1 5 10 15 Asp Val His Leu Thr Thr Val Val Gly Arg IleThr Glu Val Val Gly 20 25 30 Met Leu Ile Lys Ala Val Val Pro Asn Val ArgVal Gly Glu Val Cys 35 40 45 Leu Val Lys Arg Tyr Gly Met Glu Pro Leu ValThr Glu Val Val Gly 50 55 60 Phe Thr Gln Asn Phe Ala Phe Leu Ser Pro LeuGly Glu Leu Thr Gly 65 70 75 80 Val Ser Pro Ser Ser Glu Val Ile Pro ThrGly Leu Pro Leu Tyr Ile 85 90 95 Arg Ala Gly Asn Gly Leu Leu Gly Arg ValLeu Asn Gly Leu Gly Glu 100 105 110 Pro Ile Asp Ser Glu Ile Lys Gly ProLeu Val Asp Val Asn Glu Thr 115 120 125 Tyr Pro Val Phe Arg Ala Pro ProAsp Pro Leu His Arg Glu Lys Leu 130 135 140 Arg Thr Ile Leu Ser Thr GlyVal Arg Cys Ile Asp Gly Met Leu Thr 145 150 155 160 Val Ala Arg Gly GlnArg Ile Gly Ile Phe Ala Gly Ala Gly Val Gly 165 170 175 Lys Ser Ser LeuLeu Gly Met Ile Ala Arg Asn Ala Glu Glu Ala Asp 180 185 190 Val Asn ValIle Ala Leu Ile Gly Glu Arg Gly Arg Glu Val Arg Glu 195 200 205 Phe IleGlu Gly Asp Leu Gly Glu Glu Gly Met Lys Arg Ser Val Ile 210 215 220 ValVal Ser Thr Ser Asp Gln Ser Ser Gln Leu Arg Leu Asn Ala Ala 225 230 235240 Tyr Val Gly Thr Ala Ile Ala Glu Tyr Phe Arg Asp Gln Gly Lys Thr 245250 255 Val Val Leu Met Met Asp Ser Val Thr Arg Phe Ala Arg Ala Leu Arg260 265 270 Glu Val Gly Leu Ala Ala Gly Glu Pro Pro Ala Arg Gly Gly TyrThr 275 280 285 Pro Ser Val Phe Ser Thr Leu Pro Arg Leu Leu Glu Arg SerGly Ala 290 295 300 Ser Asp Lys Gly Thr Ile Thr Ala Phe Tyr Thr Val LeuVal Ala Gly 305 310 315 320 Asp Asp Met Asn Glu Pro Val Ala Asp Glu ValLys Ser Ile Leu Asp 325 330 335 Gly His Val Val Leu Ser Asn Ala Leu AlaGln Ala Tyr His Tyr Pro 340 345 350 Ala Ile Asp Val Leu Ala Ser Ile SerArg Leu Leu Thr Ala Ile Val 355 360 365 Pro Glu Glu Gln Arg Arg Ile IleGly Lys Ala Arg Glu Val Leu Ala 370 375 380 Lys Tyr Lys Ala Asn Glu MetLeu Ile Arg Ile Gly Glu Tyr Arg Arg 385 390 395 400 Gly Ser Asp Arg GluVal Asp Phe Ala Ile Asp His Ile Asp Lys Leu 405 410 415 Asn Arg Phe LeuLys Gln Asp Ile His Glu Lys Thr Asn Tyr Glu Glu 420 425 430 Ala Ser GlnGln Leu Arg Ala Ile Phe Arg 435 440 48 477 DNA Chlamydia psittaci 48cttcttgcag atgccgactc tgtcaacctt gcaactggat tcaacggctc cactagtgaa 60actttcaatg ttaaacaaac agataatgct gacgggacaa catatattct aggcagcgcg 120atcacctttg aacacataaa tcaattaaaa ccagcaaaca ctagctgttt tgctaataca 180gctggagatc taacgtttac tgggaatcga cgacttctct atttcaataa tatttcatca 240acagcgaaag gtgccgctat cagcacaact gcggatggta agacactcac aatatccggg 300gctctacaac tgattttcta catgtcgcca agattggcca cgggaaatgg cgtcatttat 360tctaatagct ctgtactcat cgagaacaat tctcaaggta gctcgggact gaataagtct 420gcagggaaag gcgtctttat ttgttgtgag aaaagtacgg atgtgggagc tacatca 477 49159 PRT Chlamydia psittaci 49 Leu Leu Ala Asp Ala Asp Ser Val Asn LeuAla Thr Gly Phe Asn Gly 1 5 10 15 Ser Thr Ser Glu Thr Phe Asn Val LysGln Thr Asp Asn Ala Asp Gly 20 25 30 Thr Thr Tyr Ile Leu Gly Ser Ala IleThr Phe Glu His Ile Asn Gln 35 40 45 Leu Lys Pro Ala Asn Thr Ser Cys PheAla Asn Thr Ala Gly Asp Leu 50 55 60 Thr Phe Thr Gly Asn Arg Arg Leu LeuTyr Phe Asn Asn Ile Ser Ser 65 70 75 80 Thr Ala Lys Gly Ala Ala Ile SerThr Thr Ala Asp Gly Lys Thr Leu 85 90 95 Thr Ile Ser Gly Ala Leu Gln LeuIle Phe Tyr Met Ser Pro Arg Leu 100 105 110 Ala Thr Gly Asn Gly Val IleTyr Ser Asn Ser Ser Val Leu Ile Glu 115 120 125 Asn Asn Ser Gln Gly SerSer Gly Leu Asn Lys Ser Ala Gly Lys Gly 130 135 140 Val Phe Ile Cys CysGlu Lys Ser Thr Asp Val Gly Ala Thr Ser 145 150 155 50 591 DNA Chlamydiapsittaci 50 acctttgaac acataaatca attaaaacca gcaaacacta gctgttttgctaatacagct 60 ggagatctaa cgtttactgg gaatcgacga cttctctatt tcaataatatttcatcaaca 120 gcgaaaggtg ccgctatcag cacaactgcg gatggtaaga cactcacaatatccggggct 180 ctacaactga ttttctacat gtcgccaaga ttggccacgg gaaatggcgtcatttattct 240 aatagctctg tactcatcga gaacaattct caaggtagct cgggactgaataagtctgca 300 gggaaaggcg tctttatttg ttgtgagaaa agtacggatg tgggagctacatcaccgaca 360 ttaatcatac ggaataacgg agagtttctt actgtaggta atgcagctactagctctgga 420 ggagcgattt atgcggagaa aatgatctta tcctcaggag gatatacaaaatttcaatcc 480 aatgttagct atgatcaagg tggggccatt gccattgctc ctaatggagaaattagtctc 540 tccgcggata aaggaaatat cgtctttgaa agaaacctta aaattgccaa c591 51 197 PRT Chlamydia psittaci 51 Thr Phe Glu His Ile Asn Gln Leu LysPro Ala Asn Thr Ser Cys Phe 1 5 10 15 Ala Asn Thr Ala Gly Asp Leu ThrPhe Thr Gly Asn Arg Arg Leu Leu 20 25 30 Tyr Phe Asn Asn Ile Ser Ser ThrAla Lys Gly Ala Ala Ile Ser Thr 35 40 45 Thr Ala Asp Gly Lys Thr Leu ThrIle Ser Gly Ala Leu Gln Leu Ile 50 55 60 Phe Tyr Met Ser Pro Arg Leu AlaThr Gly Asn Gly Val Ile Tyr Ser 65 70 75 80 Asn Ser Ser Val Leu Ile GluAsn Asn Ser Gln Gly Ser Ser Gly Leu 85 90 95 Asn Lys Ser Ala Gly Lys GlyVal Phe Ile Cys Cys Glu Lys Ser Thr 100 105 110 Asp Val Gly Ala Thr SerPro Thr Leu Ile Ile Arg Asn Asn Gly Glu 115 120 125 Phe Leu Thr Val GlyAsn Ala Ala Thr Ser Ser Gly Gly Ala Ile Tyr 130 135 140 Ala Glu Lys MetIle Leu Ser Ser Gly Gly Tyr Thr Lys Phe Gln Ser 145 150 155 160 Asn ValSer Tyr Asp Gln Gly Gly Ala Ile Ala Ile Ala Pro Asn Gly 165 170 175 GluIle Ser Leu Ser Ala Asp Lys Gly Asn Ile Val Phe Glu Arg Asn 180 185 190Leu Lys Ile Ala Asn 195 52 2040 DNA Chlamydia psittaci 52 atgcagggaatactaatgaa aaactctatt tatggggttt tactgttttc ctcttttgcc 60 ttatccactgctaccaaact tcttgcagat gccgactctg tcaaccttgc aactggattc 120 aacggctccactagtgaaac tttcaatgtt aaacaaacag ataatgctga cgggacaaca 180 tatattctaggcagcgcgat cacctttgaa cacataaatc aattaaaacc agcaaacact 240 agctgttttgctaatacagc tggagatcta acgtttactg ggaatcgacg acttctctat 300 ttcaataatatttcatcaac agcgaaaggt gccgctatca gcacaactgc ggatggtaag 360 acactcacaatatccggggc tctacaactg attttctaca tgtcgccaag attggccacg 420 ggaaatggcgtcatttattc taatagctct gtactcatcg agaacaattc tcaaggtagc 480 tcgggactgaataagtctgc agggaaaggc gtctttattt gttgtgagaa aagtacggat 540 gtgggagctacatcaccgac attaatcata cggaataacg gagagtttct tactgtaggt 600 aatgcagctactagctctgg aggagcgatt tatgcggaga aaatgatctt atcctcagga 660 ggatatacaaaatttcaatc caatgttagc tatgatcaag gtggggccat tgccattgct 720 cctaatggagaaattagtct ctccgcggat aaaggaaata tcgtctttga aagaaacctt 780 aaaattgccaacaaacaaaa tactcccaat gccattcacc taggagacaa tgcgaaattt 840 cttcaattacgtgctgctaa caacaaagcc atattttttt atgacccgat tacaaccacg 900 ggatctgtggcagatcggct aattattaat aactcgcaag gagaagcctc gacttacgat 960 ggggcgattgtattttctag tctcaactta ttcactcatt cccctgaatg taaactctct 1020 tcattttctcaaggtcttac tttagcggca ggatcattag ttttagaaga gggggtatgt 1080 gtacaagctccgtcttttga tcaacgtgct cactcccaac tattcatgaa tcctgggacg 1140 aagttacaagctacccagaa catctcggta aagaatctcc atctcaatct taatagaata 1200 gcagaagagccggcgtatat caccacaaca gacgatgctt ctagtgtgga catttgcgga 1260 cctgtagttatgcatataga tgatgagatc ttctataatc agacagtatt agcaaatgag 1320 ttgtctgtagagtgtttaaa tctcagttct ccacatctcg ataatatcac tattgatgac 1380 gttcccgcagtgcctatcat gacgttagaa tcgcatcgtg gttatcaagg tacatgggaa 1440 atctcttggaaagagcaacc taaacttacc tttgggaagg cgactatcgc gcctaataag 1500 cagatgcaccttatttggaa accttctggt tacgttcctt tctcaggggg aactggagag 1560 tttacgacatctttagtgcc taatagctta tggaatctct ttttagatac acgtttttct 1620 caacaagcgattgagaaaca tgctgtatct tcaggtaacg gtatatggat ttcctccatg 1680 accaattcttttcttcaagg ttctacgaac aacaaccacg gctttcgtca taagagttca 1740 ggatataccgcagggggaaa aatacaaaca cttcaagatg atatctttag tgtcagtttt 1800 tctcagctatttgggagatc taaggatttt ggatctgcca catctaagga tacattccta 1860 tcgggctctatctatgctca gcattcgaga cgcttacttc ctataatgag attccttgca 1920 ggaacatcaacatatagacc gcgactctta ctgagtattc ccaagaatct tcctatcaat 1980 tttgatgttcttgtgagtta cagctatgac agtaaccaca tgaaagtaca aaaattctaa 2040 53 679 PRTChlamydia psittaci 53 Met Gln Gly Ile Leu Met Lys Asn Ser Ile Tyr GlyVal Leu Leu Phe 1 5 10 15 Ser Ser Phe Ala Leu Ser Thr Ala Thr Lys LeuLeu Ala Asp Ala Asp 20 25 30 Ser Val Asn Leu Ala Thr Gly Phe Asn Gly SerThr Ser Glu Thr Phe 35 40 45 Asn Val Lys Gln Thr Asp Asn Ala Asp Gly ThrThr Tyr Ile Leu Gly 50 55 60 Ser Ala Ile Thr Phe Glu His Ile Asn Gln LeuLys Pro Ala Asn Thr 65 70 75 80 Ser Cys Phe Ala Asn Thr Ala Gly Asp LeuThr Phe Thr Gly Asn Arg 85 90 95 Arg Leu Leu Tyr Phe Asn Asn Ile Ser SerThr Ala Lys Gly Ala Ala 100 105 110 Ile Ser Thr Thr Ala Asp Gly Lys ThrLeu Thr Ile Ser Gly Ala Leu 115 120 125 Gln Leu Ile Phe Tyr Met Ser ProArg Leu Ala Thr Gly Asn Gly Val 130 135 140 Ile Tyr Ser Asn Ser Ser ValLeu Ile Glu Asn Asn Ser Gln Gly Ser 145 150 155 160 Ser Gly Leu Asn LysSer Ala Gly Lys Gly Val Phe Ile Cys Cys Glu 165 170 175 Lys Ser Thr AspVal Gly Ala Thr Ser Pro Thr Leu Ile Ile Arg Asn 180 185 190 Asn Gly GluPhe Leu Thr Val Gly Asn Ala Ala Thr Ser Ser Gly Gly 195 200 205 Ala IleTyr Ala Glu Lys Met Ile Leu Ser Ser Gly Gly Tyr Thr Lys 210 215 220 PheGln Ser Asn Val Ser Tyr Asp Gln Gly Gly Ala Ile Ala Ile Ala 225 230 235240 Pro Asn Gly Glu Ile Ser Leu Ser Ala Asp Lys Gly Asn Ile Val Phe 245250 255 Glu Arg Asn Leu Lys Ile Ala Asn Lys Gln Asn Thr Pro Asn Ala Ile260 265 270 His Leu Gly Asp Asn Ala Lys Phe Leu Gln Leu Arg Ala Ala AsnAsn 275 280 285 Lys Ala Ile Phe Phe Tyr Asp Pro Ile Thr Thr Thr Gly SerVal Ala 290 295 300 Asp Arg Leu Ile Ile Asn Asn Ser Gln Gly Glu Ala SerThr Tyr Asp 305 310 315 320 Gly Ala Ile Val Phe Ser Ser Leu Asn Leu PheThr His Ser Pro Glu 325 330 335 Cys Lys Leu Ser Ser Phe Ser Gln Gly LeuThr Leu Ala Ala Gly Ser 340 345 350 Leu Val Leu Glu Glu Gly Val Cys ValGln Ala Pro Ser Phe Asp Gln 355 360 365 Arg Ala His Ser Gln Leu Phe MetAsn Pro Gly Thr Lys Leu Gln Ala 370 375 380 Thr Gln Asn Ile Ser Val LysAsn Leu His Leu Asn Leu Asn Arg Ile 385 390 395 400 Ala Glu Glu Pro AlaTyr Ile Thr Thr Thr Asp Asp Ala Ser Ser Val 405 410 415 Asp Ile Cys GlyPro Val Val Met His Ile Asp Asp Glu Ile Phe Tyr 420 425 430 Asn Gln ThrVal Leu Ala Asn Glu Leu Ser Val Glu Cys Leu Asn Leu 435 440 445 Ser SerPro His Leu Asp Asn Ile Thr Ile Asp Asp Val Pro Ala Val 450 455 460 ProIle Met Thr Leu Glu Ser His Arg Gly Tyr Gln Gly Thr Trp Glu 465 470 475480 Ile Ser Trp Lys Glu Gln Pro Lys Leu Thr Phe Gly Lys Ala Thr Ile 485490 495 Ala Pro Asn Lys Gln Met His Leu Ile Trp Lys Pro Ser Gly Tyr Val500 505 510 Pro Phe Ser Gly Gly Thr Gly Glu Phe Thr Thr Ser Leu Val ProAsn 515 520 525 Ser Leu Trp Asn Leu Phe Leu Asp Thr Arg Phe Ser Gln GlnAla Ile 530 535 540 Glu Lys His Ala Val Ser Ser Gly Asn Gly Ile Trp IleSer Ser Met 545 550 555 560 Thr Asn Ser Phe Leu Gln Gly Ser Thr Asn AsnAsn His Gly Phe Arg 565 570 575 His Lys Ser Ser Gly Tyr Thr Ala Gly GlyLys Ile Gln Thr Leu Gln 580 585 590 Asp Asp Ile Phe Ser Val Ser Phe SerGln Leu Phe Gly Arg Ser Lys 595 600 605 Asp Phe Gly Ser Ala Thr Ser LysAsp Thr Phe Leu Ser Gly Ser Ile 610 615 620 Tyr Ala Gln His Ser Arg ArgLeu Leu Pro Ile Met Arg Phe Leu Ala 625 630 635 640 Gly Thr Ser Thr TyrArg Pro Arg Leu Leu Leu Ser Ile Pro Lys Asn 645 650 655 Leu Pro Ile AsnPhe Asp Val Leu Val Ser Tyr Ser Tyr Asp Ser Asn 660 665 670 His Met LysVal Gln Lys Phe 675 54 487 DNA Chlamydia psittaci 54 acctcgagagaggattctct tagtgtggct ttctgtcagt tatttgcaaa agataaagac 60 taccttgtaagcaagaacgc cgcaaacgtc tatgcgggtt ctgtatatta tcagcatgtg 120 agcaagtttgatgatctcac gcggttattt aatgggccta acacgtgttg ttcagggttt 180 tctaaagagattcctatttt cttggatgca caaattacct attgccacac ggccaacaac 240 atgacaacgtcctatacaga ctatcctgaa gtgaaaggtt cttggggtaa tgataccctg 300 ggcttaactttgtctactag cgtacctatc ccggtattta gttcttctat ctttgatagt 360 tatgcaccgtttgcaaaatt acaagttgtc tatgcgcacc aagatgactt taaagaacca 420 acaacagaaggccgggtctt tgaaagcagc gatcttctca acgtttctgt acctataggt 480 ataaaat 48755 162 PRT Chlamydia psittaci 55 Thr Ser Arg Glu Asp Ser Leu Ser Val AlaPhe Cys Gln Leu Phe Ala 1 5 10 15 Lys Asp Lys Asp Tyr Leu Val Ser LysAsn Ala Ala Asn Val Tyr Ala 20 25 30 Gly Ser Val Tyr Tyr Gln His Val SerLys Phe Asp Asp Leu Thr Arg 35 40 45 Leu Phe Asn Gly Pro Asn Thr Cys CysSer Gly Phe Ser Lys Glu Ile 50 55 60 Pro Ile Phe Leu Asp Ala Gln Ile ThrTyr Cys His Thr Ala Asn Asn 65 70 75 80 Met Thr Thr Ser Tyr Thr Asp TyrPro Glu Val Lys Gly Ser Trp Gly 85 90 95 Asn Asp Thr Leu Gly Leu Thr LeuSer Thr Ser Val Pro Ile Pro Val 100 105 110 Phe Ser Ser Ser Ile Phe AspSer Tyr Ala Pro Phe Ala Lys Leu Gln 115 120 125 Val Val Tyr Ala His GlnAsp Asp Phe Lys Glu Pro Thr Thr Glu Gly 130 135 140 Arg Val Phe Glu SerSer Asp Leu Leu Asn Val Ser Val Pro Ile Gly 145 150 155 160 Ile Lys 562781 DNA Chlamydia psittaci 56 atgaggcctt ctttatataa gattttaatatcgtcgacgc tgacgttacc aatatctttt 60 cacttctcgc aattgcatgc agaagtggctttaactcaag aatctattct cgatgcaaat 120 ggagcattca gtccgcaatc tacaagcactgcgggaggaa cgatttacaa cgtcgagagt 180 gatatttcta ttgtagatgt aggacagacagcggctcttg cttcctcagc ttttgttcag 240 actgcagaca acctaacttt caaagggaacaaccatagct tatccataac gaacgcgaat 300 gccggagcta atcctgcggg aattaacgttaacactgccg ataagattct tacgctgaca 360 gatttttcta agttgagctt taaggaatgcccatcttctc tagtgaatac tggaaaaggg 420 gctatgaaat ccggaggagc attaaacttagcgaataatg ccagtattct gtttgatcag 480 aactattccg ctgagaatgg tggagccatctcttgcaaag ctttttctct aaccggctcg 540 agcaaagaaa tcagcttcac cactaactctactgcgaaaa aaggtggagc gattgctgct 600 acgggaatag ctcatctttc ggacaaccaaggcacaatca gattttctgg gaacactgct 660 gtgaattctg ggggagcagt atattcagaagcttctatga cgattgcagg taacaaccac 720 gttgctttta gcaacaatgc tgtttccggttcatctgatg gttgcggtgg agctatccat 780 tgtagcaaaa caggttcagc accgacccttactataagag ataacaaagt cttgattttt 840 gaggaaaata cttcttcagc aaaaggtggagcgatttaca ccgataaact catattgact 900 tctggtgggc ctacggcatt tatcaataacaaagttaccc atgctacacc taagggtgga 960 gctattggta ttgctgccaa tggagaatgtagcttaaccg ctgaacatgg ggatattact 1020 tttgataata acctgatggc cacacaagacaatgctacaa taaaaagaaa tgccattaac 1080 attgaaggca atggtaaatt cgtcaacttacgtgcagcgt ctggaaagac gatttctttc 1140 tatgatccta tcacagttga aggtaatgctgctgatcttc tcactttgaa taaagctgag 1200 ggtgataaaa cgtataatgg aagaattattttttcaggag aaaagctcac tgaagaacaa 1260 gctgctgttg cggataacct aaagacaacatttacacagc ctatcacttt agctgctggt 1320 gaacttgtgt tacgcagcgg tgtggaagtagaagcaaaaa cagtcgtgca aacagcagga 1380 tctttgattc tgatggatgc aggcacaaagttatccgcaa aaacagaaga tgctacactg 1440 acgaatctgg ctattaatcc gaataccttagatgggaaaa aattcgccgt agtcgatgcc 1500 gttgctgctg ggaagaatgt gactttatcaggtgctattg gcgttattga tcctacaggg 1560 aagttttatg aaaaccataa gctaaatgatacgttagctt taggaggaat tcaactttct 1620 gggaaaggtt cggtgacaac aaccaacgtgcctagtcatg ttgttggtgt tgctgaaacc 1680 cactatggtt atcaaggaaa ctggtctgtcagttgggtca aagataataa ctctgatcct 1740 aaaacacaaa cagcaatctt tacctggaataaaacaggat atgttccaaa tcctgaacgt 1800 cgtgctccgc tagtactcaa tagcctttggggatccttta tagatttacg ttctattcaa 1860 gatgtcttgg aacgtagtgt tgatagtattcttgagacac gtcgtggttt gtgggtctct 1920 ggaattggga acttcttcca taaagatcggaatgctgaaa atcgcaaatt ccgtcatatc 1980 agttcgggat atgtgttagg agccacaacaaatacctcga gagaggattc tcttagtgtg 2040 gctttctgtc agttatttgc aaaagataaagactaccttg taagcaagaa cgccgcaaac 2100 gtctatgcgg gttctgtata ttatcagcatgtgagcaagt ttgatgatct cacgcggtta 2160 tttaatgggc ctaacacgtg ttgttcagggttttctaaag agattcctat tttcttggat 2220 gcacaaatta cctattgcca cacggccaacaacatgacaa cgtcctatac agactatcct 2280 gaagtgaaag gttcttgggg taatgataccctgggcttaa ctttgtctac tagcgtacct 2340 atcccggtat ttagttcttc tatctttgatagttatgcac cgtttgcaaa attacaagtt 2400 gtctatgcgc accaagatga ctttaaagaaccaacaacag aaggccgggt ctttgaaagc 2460 agcgatcttc tcaacgtttc tgtacctataggtataaaat ttgagaaact ctcctatgga 2520 gagagaagtg cttatgatct tacactgatgtatatacctg atgtgtaccg tcataatcca 2580 agctgtatga caggattggc gatcaatgacgtttcctggt taaccacagc tacgaatctt 2640 gctagacaag ctttcatagt tcgcgcgggtaaccatattg ccttaacctc tggtgttgag 2700 atgttcagtc agtttggttt cgaattacgaagctcttcaa gaaattataa cgtagatctt 2760 ggcgctaagg tcgcgttcta a 2781 57926 PRT Chlamydia psittaci 57 Met Arg Pro Ser Leu Tyr Lys Ile Leu IleSer Ser Thr Leu Thr Leu 1 5 10 15 Pro Ile Ser Phe His Phe Ser Gln LeuHis Ala Glu Val Ala Leu Thr 20 25 30 Gln Glu Ser Ile Leu Asp Ala Asn GlyAla Phe Ser Pro Gln Ser Thr 35 40 45 Ser Thr Ala Gly Gly Thr Ile Tyr AsnVal Glu Ser Asp Ile Ser Ile 50 55 60 Val Asp Val Gly Gln Thr Ala Ala LeuAla Ser Ser Ala Phe Val Gln 65 70 75 80 Thr Ala Asp Asn Leu Thr Phe LysGly Asn Asn His Ser Leu Ser Ile 85 90 95 Thr Asn Ala Asn Ala Gly Ala AsnPro Ala Gly Ile Asn Val Asn Thr 100 105 110 Ala Asp Lys Ile Leu Thr LeuThr Asp Phe Ser Lys Leu Ser Phe Lys 115 120 125 Glu Cys Pro Ser Ser LeuVal Asn Thr Gly Lys Gly Ala Met Lys Ser 130 135 140 Gly Gly Ala Leu AsnLeu Ala Asn Asn Ala Ser Ile Leu Phe Asp Gln 145 150 155 160 Asn Tyr SerAla Glu Asn Gly Gly Ala Ile Ser Cys Lys Ala Phe Ser 165 170 175 Leu ThrGly Ser Ser Lys Glu Ile Ser Phe Thr Thr Asn Ser Thr Ala 180 185 190 LysLys Gly Gly Ala Ile Ala Ala Thr Gly Ile Ala His Leu Ser Asp 195 200 205Asn Gln Gly Thr Ile Arg Phe Ser Gly Asn Thr Ala Val Asn Ser Gly 210 215220 Gly Ala Val Tyr Ser Glu Ala Ser Met Thr Ile Ala Gly Asn Asn His 225230 235 240 Val Ala Phe Ser Asn Asn Ala Val Ser Gly Ser Ser Asp Gly CysGly 245 250 255 Gly Ala Ile His Cys Ser Lys Thr Gly Ser Ala Pro Thr LeuThr Ile 260 265 270 Arg Asp Asn Lys Val Leu Ile Phe Glu Glu Asn Thr SerSer Ala Lys 275 280 285 Gly Gly Ala Ile Tyr Thr Asp Lys Leu Ile Leu ThrSer Gly Gly Pro 290 295 300 Thr Ala Phe Ile Asn Asn Lys Val Thr His AlaThr Pro Lys Gly Gly 305 310 315 320 Ala Ile Gly Ile Ala Ala Asn Gly GluCys Ser Leu Thr Ala Glu His 325 330 335 Gly Asp Ile Thr Phe Asp Asn AsnLeu Met Ala Thr Gln Asp Asn Ala 340 345 350 Thr Ile Lys Arg Asn Ala IleAsn Ile Glu Gly Asn Gly Lys Phe Val 355 360 365 Asn Leu Arg Ala Ala SerGly Lys Thr Ile Ser Phe Tyr Asp Pro Ile 370 375 380 Thr Val Glu Gly AsnAla Ala Asp Leu Leu Thr Leu Asn Lys Ala Glu 385 390 395 400 Gly Asp LysThr Tyr Asn Gly Arg Ile Ile Phe Ser Gly Glu Lys Leu 405 410 415 Thr GluGlu Gln Ala Ala Val Ala Asp Asn Leu Lys Thr Thr Phe Thr 420 425 430 GlnPro Ile Thr Leu Ala Ala Gly Glu Leu Val Leu Arg Ser Gly Val 435 440 445Glu Val Glu Ala Lys Thr Val Val Gln Thr Ala Gly Ser Leu Ile Leu 450 455460 Met Asp Ala Gly Thr Lys Leu Ser Ala Lys Thr Glu Asp Ala Thr Leu 465470 475 480 Thr Asn Leu Ala Ile Asn Pro Asn Thr Leu Asp Gly Lys Lys PheAla 485 490 495 Val Val Asp Ala Val Ala Ala Gly Lys Asn Val Thr Leu SerGly Ala 500 505 510 Ile Gly Val Ile Asp Pro Thr Gly Lys Phe Tyr Glu AsnHis Lys Leu 515 520 525 Asn Asp Thr Leu Ala Leu Gly Gly Ile Gln Leu SerGly Lys Gly Ser 530 535 540 Val Thr Thr Thr Asn Val Pro Ser His Val ValGly Val Ala Glu Thr 545 550 555 560 His Tyr Gly Tyr Gln Gly Asn Trp SerVal Ser Trp Val Lys Asp Asn 565 570 575 Asn Ser Asp Pro Lys Thr Gln ThrAla Ile Phe Thr Trp Asn Lys Thr 580 585 590 Gly Tyr Val Pro Asn Pro GluArg Arg Ala Pro Leu Val Leu Asn Ser 595 600 605 Leu Trp Gly Ser Phe IleAsp Leu Arg Ser Ile Gln Asp Val Leu Glu 610 615 620 Arg Ser Val Asp SerIle Leu Glu Thr Arg Arg Gly Leu Trp Val Ser 625 630 635 640 Gly Ile GlyAsn Phe Phe His Lys Asp Arg Asn Ala Glu Asn Arg Lys 645 650 655 Phe ArgHis Ile Ser Ser Gly Tyr Val Leu Gly Ala Thr Thr Asn Thr 660 665 670 SerArg Glu Asp Ser Leu Ser Val Ala Phe Cys Gln Leu Phe Ala Lys 675 680 685Asp Lys Asp Tyr Leu Val Ser Lys Asn Ala Ala Asn Val Tyr Ala Gly 690 695700 Ser Val Tyr Tyr Gln His Val Ser Lys Phe Asp Asp Leu Thr Arg Leu 705710 715 720 Phe Asn Gly Pro Asn Thr Cys Cys Ser Gly Phe Ser Lys Glu IlePro 725 730 735 Ile Phe Leu Asp Ala Gln Ile Thr Tyr Cys His Thr Ala AsnAsn Met 740 745 750 Thr Thr Ser Tyr Thr Asp Tyr Pro Glu Val Lys Gly SerTrp Gly Asn 755 760 765 Asp Thr Leu Gly Leu Thr Leu Ser Thr Ser Val ProIle Pro Val Phe 770 775 780 Ser Ser Ser Ile Phe Asp Ser Tyr Ala Pro PheAla Lys Leu Gln Val 785 790 795 800 Val Tyr Ala His Gln Asp Asp Phe LysGlu Pro Thr Thr Glu Gly Arg 805 810 815 Val Phe Glu Ser Ser Asp Leu LeuAsn Val Ser Val Pro Ile Gly Ile 820 825 830 Lys Phe Glu Lys Leu Ser TyrGly Glu Arg Ser Ala Tyr Asp Leu Thr 835 840 845 Leu Met Tyr Ile Pro AspVal Tyr Arg His Asn Pro Ser Cys Met Thr 850 855 860 Gly Leu Ala Ile AsnAsp Val Ser Trp Leu Thr Thr Ala Thr Asn Leu 865 870 875 880 Ala Arg GlnAla Phe Ile Val Arg Ala Gly Asn His Ile Ala Leu Thr 885 890 895 Ser GlyVal Glu Met Phe Ser Gln Phe Gly Phe Glu Leu Arg Ser Ser 900 905 910 SerArg Asn Tyr Asn Val Asp Leu Gly Ala Lys Val Ala Phe 915 920 925 58 559DNA Chlamydia psittaci 58 tgtgttcatt ctttagcagg agttgcattt acgttgtttctctgtgagca tatgtttacc 60 aatatgcttg cttcttctta ttttaaggaa ggcagtggttttgttcagtt agtgagcaaa 120 tttcatcaga ttcctggtct gaagatcata gaaattgtttttttagccct accgtttact 180 tgtcacgcta tcctaggtat tttctatctt tttcaagcgcaaactaattc acgggcttct 240 gacggcagaa aacccgcgtt aatctatgcg agaaatcttgcctatacttg gcagagaaga 300 actgcttgga ttttactttt cggtcttatt tttcacgtagttcagtttcg ttttcttcgt 360 tatcctattc atgtagagct gcatgggcaa acatactatgttgtcgatat tgacgcttct 420 cggtatgcgg cgatagtgcg gggtacacaa ggattttttactataaattt ttcagctcct 480 caacttgaaa cgattcgttt ggataaagag gatcttgacggcagcgcagt ttctcaatta 540 ttagacagaa aagcgtatc 559 59 186 PRT Chlamydiapsittaci 59 Cys Val His Ser Leu Ala Gly Val Ala Phe Thr Leu Phe Leu CysGlu 1 5 10 15 His Met Phe Thr Asn Met Leu Ala Ser Ser Tyr Phe Lys GluGly Ser 20 25 30 Gly Phe Val Gln Leu Val Ser Lys Phe His Gln Ile Pro GlyLeu Lys 35 40 45 Ile Ile Glu Ile Val Phe Leu Ala Leu Pro Phe Thr Cys HisAla Ile 50 55 60 Leu Gly Ile Phe Tyr Leu Phe Gln Ala Gln Thr Asn Ser ArgAla Ser 65 70 75 80 Asp Gly Arg Lys Pro Ala Leu Ile Tyr Ala Arg Asn LeuAla Tyr Thr 85 90 95 Trp Gln Arg Arg Thr Ala Trp Ile Leu Leu Phe Gly LeuIle Phe His 100 105 110 Val Val Gln Phe Arg Phe Leu Arg Tyr Pro Ile HisVal Glu Leu His 115 120 125 Gly Gln Thr Tyr Tyr Val Val Asp Ile Asp AlaSer Arg Tyr Ala Ala 130 135 140 Ile Val Arg Gly Thr Gln Gly Phe Phe ThrIle Asn Phe Ser Ala Pro 145 150 155 160 Gln Leu Glu Thr Ile Arg Leu AspLys Glu Asp Leu Asp Gly Ser Ala 165 170 175 Val Ser Gln Leu Leu Asp ArgLys Ala Tyr 180 185 60 687 DNA Chlamydia psittaci 60 atgatgaatgaaaaggaatc atgttctgag gctactcaga ggtcatggaa gtactacact 60 agctttgttttacgttgtgt tcattcttta gcaggagttg catttacgtt gtttctctgt 120 gagcatatgtttaccaatat gcttgcttct tcttatttta aggaaggcag tggttttgtt 180 cagttagtgagcaaatttca tcagattcct ggtctgaaga tcatagaaat tgttttttta 240 gccctaccgtttacttgtca cgctatccta ggtattttct atctttttca agcgcaaact 300 aattcacgggcttctgacgg cagaaaaccc gcgttaatct atgcgagaaa tcttgcctat 360 acttggcagagaagaactgc ttggatttta cttttcggtc ttatttttca cgtagttcag 420 tttcgttttcttcgttatcc tattcatgta gagctgcatg ggcaaacata ctatgttgtc 480 gatattgacgcttctcggta tgcggcgata gtgcggggta cacaaggatt ttttactata 540 aatttttcagctcctcaact tgaaacgatt cgtttggata aagaggatct tgacggcagc 600 gcagtttctcaattattaga cagaaaagcg tatctcctga ctcctaatgt tggaccgctt 660 ttctttatgttgttcgggat tctttag 687 61 228 PRT Chlamydia psittaci 61 Met Met Asn GluLys Glu Ser Cys Ser Glu Ala Thr Gln Arg Ser Trp 1 5 10 15 Lys Tyr TyrThr Ser Phe Val Leu Arg Cys Val His Ser Leu Ala Gly 20 25 30 Val Ala PheThr Leu Phe Leu Cys Glu His Met Phe Thr Asn Met Leu 35 40 45 Ala Ser SerTyr Phe Lys Glu Gly Ser Gly Phe Val Gln Leu Val Ser 50 55 60 Lys Phe HisGln Ile Pro Gly Leu Lys Ile Ile Glu Ile Val Phe Leu 65 70 75 80 Ala LeuPro Phe Thr Cys His Ala Ile Leu Gly Ile Phe Tyr Leu Phe 85 90 95 Gln AlaGln Thr Asn Ser Arg Ala Ser Asp Gly Arg Lys Pro Ala Leu 100 105 110 IleTyr Ala Arg Asn Leu Ala Tyr Thr Trp Gln Arg Arg Thr Ala Trp 115 120 125Ile Leu Leu Phe Gly Leu Ile Phe His Val Val Gln Phe Arg Phe Leu 130 135140 Arg Tyr Pro Ile His Val Glu Leu His Gly Gln Thr Tyr Tyr Val Val 145150 155 160 Asp Ile Asp Ala Ser Arg Tyr Ala Ala Ile Val Arg Gly Thr GlnGly 165 170 175 Phe Phe Thr Ile Asn Phe Ser Ala Pro Gln Leu Glu Thr IleArg Leu 180 185 190 Asp Lys Glu Asp Leu Asp Gly Ser Ala Val Ser Gln LeuLeu Asp Arg 195 200 205 Lys Ala Tyr Leu Leu Thr Pro Asn Val Gly Pro LeuPhe Phe Met Leu 210 215 220 Phe Gly Ile Leu 225

What is claimed is:
 1. An isolated polynucleotide comprising a regionhaving a sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, 5 SEQ ID NO:18, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:58, or SEQ IDNO:60, or fragment thereof.
 2. An isolated polynucleotide comprising aregion having a sequence having at least 17 contiguous nucleotides incommon with at least one of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID 15 NO:44, SEQ IDNO:46, SEQ ID NO:58, or SEQ ID NO:60, or its complement.
 3. The isolatedpolynucleotide of claim 2, further defined as comprising a sequencehaving least 50 contiguous nucleotides in common with at least one ofSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:58, or SEQ IDNO:60, or its complement.
 4. The isolated polynucleotide of claim 3,further defined as comprising a sequence having all nucleotides incommon with at least one of SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:58, or SEQ IDNO:60, or its complement.
 5. A polypeptide having a sequence of SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:59, SEQ IDNO:61 or fragment thereof.
 6. The polypeptide of claim 5, furtherdefined as a recombinant polypeptide.
 7. A method of producing apolypeptide having a sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25,SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45,SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:61 or fragment thereof,comprising: a) obtaining a polynucleotide comprising a region encodingthe sequence; and b) expressing the polynucleotide to obtain thepolypeptide.
 8. The method of claim 7, wherein the polynucleotide has aregion having a sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:58, or SEQ ID NO:60, or fragment thereof
 9. An antibody directedagainst an antigen having sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ IDNO:61, or antigenic fragment thereof.
 10. The antibody of claim 9,further defined as a monoclonal antibody.
 11. A vaccine for theimmunization of a bovine against Chlamydia psittaci comprising: (a) apharmaceutically acceptable carrier, and (b) at least one polynucleotidehaving a Chlamydia psittaci sequence.
 12. The vaccine of claim 1 1,wherein the at least one polynucleotide has a sequence isolated from aChlamydia psittaci genomic DNA expression library
 13. The vaccine ofclaim 11, wherein the at least one polynucleotide has a sequence of SEQID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO52:, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO: 58, or SEQ ID NO:60, or fragment thereof.
 14. Thevaccine of claim 13, wherein the at least one polynucleotide has asequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ IDNO:26, or fragment thereof.
 15. The vaccine of claim 13, wherein the atleast one polynucleotide has a sequence of SEQ ID NO:6, SEQ ID NO:10,SEQ ID NO:14, SEQ ID NO:20, or SEQ ID NO:24.
 16. The vaccine of claim11, wherein the polynucleotide is comprised in a genetic immunizationvector.
 17. The vaccine of claim 16, wherein the vector comprises a geneencoding a mouse ubiquitin fusion polypeptide.
 18. The vaccine of claim16, wherein the vector comprises a promoter operable in eukaryoticcells.
 19. The vaccine of claim 18, wherein the promoter is a CMVpromoter.
 20. The vaccine of claim 11, wherein the polynucleotide iscloned into a viral expression vector.
 21. The vaccine of claim 20,wherein the viral expression vector is selected from the groupconsisting of adenovirus, adeno-associated virus, retrovirus andherpes-simplex virus.
 22. The vaccine of claim 11, comprising apolynucleotide encoding a antigen having a sequence of SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, or SEQ ID NO:61, or antigenic fragment thereof.
 23. The vaccineof claim 11, comprising at least a first polynucleotide having aChlamydia psittaci sequence and a second polynucleotide having aChlamydia psittaci sequence, wherein the first polynucleotide and thesecond polynucleotide have different Chlamydia psittaci sequences. 24.The vaccine of claim 23, wherein the first polynucleotide has a sequenceof SEQ ID NO:50.
 25. A vaccine for the immunization of a bovine againstChlamydia psittaci comprising: (a) a pharmaceutically acceptablecarrier; and (b) at least one Chlamydia psittaci antigen.
 26. Thevaccine of claim 25, wherein the at least one Chlamydia psittaci antigenhas a sequence of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO=35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, or SEQ ID NO:61, or antigenicfragment thereof.
 27. The vaccine of claim 25, wherein the at least oneChlamydia psittaci antigen has a sequence of SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, or SEQ ID NO:27, or an antigenic fragmentthereof.
 28. The vaccine of claim 25, wherein the at least one Chlamydiapsittaci antigen has a sequence of SEQ ID NO:7, SEQ ID NO:11, SEQ IDNO:15, SEQ ID NO:21, or SEQ ID NO:25.
 29. A method of immunizing abovine comprising providing to the bovine at least one Chlamydiapsittaci antigen, or antigenic fragment thereof, in an amount effectiveto induce an immune response.
 30. The method of claim 29, wherein the atleast one Chlamydia psittaci antigen has a sequence of SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, or SEQ ID NO:61, or an antigenic fragment thereof.
 31. The methodof claim 29, wherein the provision of the at least one Chlamydiapsittaci antigen comprises: (a) preparing a cloned expression libraryfrom fragmented genomic DNA, cDNA or sequenced genes of Chlamydiapsittaci; (b) administering at least one clone of the library in apharmaceutically acceptable carrier into the bovine; and (c) expressingat least one Chlamydia psittaci antigen in the bovine.
 32. The method ofclaim 31, wherein the expression library comprises at least one or morepolynucleotide having a sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO52:, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, or SEQID NO:60, or fragment thereof.
 33. The method of claim 32, wherein theexpression library is cloned in a genetic immunization vector of SEQ IDNO:
 1. 34. The method of claim 33, wherein the vector comprises a geneencoding a mouse ubiquitin fusion polypeptide designed to link theexpression library polynucleotides to the ubiquitin gene.
 35. The methodof claim 34, wherein the vector comprises a promoter operable ineukaryotic cells.
 36. The method of claim 35, wherein the promoter is aCMV promoter.
 37. The method of claim 32, wherein the polynucleotide isadministered by a intramuscular injection or epidermal injection. 38.The method of claim 37, wherein the polynucleotide is administered byintravenous, subcutaneous, intralesional, intraperitoneal, oral orinhaled routes of administration.
 39. The method of claim 38, wherein asecond intramuscular injection and epidermal injection are administeredat least about three weeks after the first injection.
 40. The method ofclaim 32, wherein the polynucleotide is cloned into a viral expressionvector.
 41. The method of claim 29, wherein the provision of theChlamydia psittaci antigen(s) comprises: (a) preparing a pharmaceuticalcomposition comprising at least one polynucleotide having a sequence ofSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO52:, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO: 58, or SEQ ID NO:60, or fragment thereof; (b)administering one or more clones of the library in a pharmaceuticallyacceptable carrier into the bovine; and (c) expressing one or moreChlamydia psittaci antigens in the bovine.
 42. The method of claim 41,wherein the one or more polynucleotides is in one or more expressionvectors.
 43. The method of claim 42, wherein the one or morepolynucleotides are cloned in a genetic immunization vector of SEQ IDNO:1.
 44. The method of claim 29, wherein the provision of the Chlamydiaantigen(s) comprises: (a) preparing a pharmaceutical composition of atleast one Chlamydia antigen having a sequence of SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, or SEQ ID NO:61, or an antigenic fragment thereof; and (b)administering the at least one antigen or fragment into the bovine. 45.The method of claim 44, wherein the antigen(s) is administered by afirst intramuscular injection, intravenous injection, parenteralinjection, epidermal injection, inhalation or oral route.
 46. The methodof claim 45, wherein a second intramuscular injection, intravenousinjection, parenteral injection or epidermal injection is administeredat least about three weeks after the first injection.
 47. A method ofassaying for the presence of Chlamydia psittaci infection in a bovinecomprising: (a) obtaining an antibody directed against a Chlamydiapsittaci antigen; (b) obtaining a sample from the bovine; (c) admixingthe antibody with the sample; and (d) assaying the sample forantigen-antibody binding, wherein the antigen-antibody binding indicatesChlamydia psittaci infection in the bovine.
 48. The method of claim 47,wherein the antibody directed against the antigen is further defined asa monoclonal antibody.
 49. The method of claim 47, wherein assaying thesample for antigen-antibody binding is by precipitin reaction,radioimmunoassay, ELISA, Western blot or immunofluorescence.
 50. A kitfor assaying a Chlamydia psittaci infection comprising, in a suitablecontainer: (a) a pharmaceutically acceptable carrier; and (b) anantibody directed against a Chlamydia psittaci antigen.
 51. A method ofassaying for the presence of a Chlamydia psittaci infection in a bovinecomprising: (a) obtaining an oligonucleotide probe comprising a sequencecomprised within one of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO: 58, or SEQ ID NO:60, or acomplement thereof, and (b) employing the probe in a PCR detectionprotocol.